Organelle genome modification using polynucleotide guided endonuclease

ABSTRACT

Provided herein are methods and systems for altering the genome of an organelle. In some embodiments, the method comprises introducing into an organelle a recombinant DNA construct comprising a first polynucleotide encoding at least one guide RNA and a second polynucleotide encoding a polynucleotide guided polypeptide; and growing a cell comprising the organelle under conditions in which the first polynucleotide and the second polynucleotide are each expressed.

CROSS-REFERENCE

This application is related to U.S. Provisional Patent Application No.62/548,723, filed on Aug. 22, 2017, which is entirely incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2018, isnamed 51090_701_201 SL.txt and is 204,800 bytes in size.

SUMMARY

In an aspect, a method for altering the genome of an organelle maycomprise: (a) introducing into an organelle comprising the following:(i) a first polynucleotide encoding at least one guide polynucleic acid,wherein the at least one guide polynucleic acid directs a polynucleotideguided polypeptide to cleave at least one target sequence present in anorganelle genome; (ii) a second polynucleotide encoding a polynucleotideguided polypeptide, wherein the polynucleotide guided polypeptide, whenassociated with the guide polynucleic acid, cleaves the at least onetarget sequence; (iii) optionally, a third polynucleotide encoding atleast one homologous organelle DNA sequence, wherein the at least onehomologous organelle DNA is of sufficient size for homologousrecombination, wherein integration of the at least one homologousorganelle DNA sequence into the organelle genome results in removal ofthe at least one target sequence; (iv) optionally, a fourthpolynucleotide encoding at least one selectable marker or at least onescreenable marker, or both; wherein the fourth polynucleotide isoperably linked to a promoter that is functional in the organelle; and(v) optionally, a fifth polynucleotide encoding an origin of replicationthat is functional in the organelle; and (b) growing a cell comprisingthe organelle of (a) under conditions in which the first polynucleotideof (i) and the second polynucleotide of (ii) are each expressed.

In another aspect, a method for altering the genome of an organelle maycomprise: (a) introducing into an organelle a recombinant DNA constructcomprising the following: (i) a first polynucleotide encoding at leastone guide polynucleic acid, wherein the at least one guide polynucleicacid directs a polynucleotide guided polypeptide to cleave at least onetarget sequence present in an organelle genome; (ii) a secondpolynucleotide encoding a polynucleotide guided polypeptide, wherein thepolynucleotide guided polypeptide, when associated with the guidepolynucleic acid, cleaves the at least one target sequence; (iii)optionally, a third polynucleotide encoding at least one homologousorganelle DNA sequence, wherein the at least one homologous organelleDNA is of sufficient size for homologous recombination, whereinintegration of the at least one homologous organelle DNA sequence intothe organelle genome results in removal of the at least one targetsequence; (iv) optionally, a fourth polynucleotide encoding at least oneselectable marker or at least one screenable marker, or both; whereinthe fourth polynucleotide is operably linked to a promoter that isfunctional in the organelle; and (v) optionally, a fifth polynucleotideencoding an origin of replication that is functional in the organelle;and (b) growing a cell comprising the organelle of (a) under conditionsin which the first polynucleotide of (i) and the second polynucleotideof (ii) are each expressed

In some embodiments, the method may further comprise a step (c) ofselecting a cell having an organelle that comprises an altered genome.In some embodiments, the method may further comprise a step (d) ofselecting a cell that is homoplasmic for the altered genome of theorganelle.

In some embodiments, the method may comprise introducing into anorganelle the third polynucleotide of (iii), wherein the thirdpolynucleotide of (iii) may comprise a sixth and a seventhpolynucleotide, wherein the sixth and the seventh polynucleotidescorrespond to two adjacent regions of homology in the organelle genome,wherein the sixth and seventh polynucleotides are separated by asequence that is heterologous to the organelle DNA. In some embodiments,the sequence that is heterologous to the organelle DNA may comprise atleast one selected from the group consisting of: the firstpolynucleotide, the second polynucleotide, the fourth polynucleotide, aneighth polynucleotide, and any combination thereof, wherein the eighthpolynucleotide encodes an RNA that is heterologous to the organelle.

In another embodiment, the at least one guide polynucleic acid may bepresent on a polycistronic transcription unit. In some embodiments, theat least one guide polynucleic acid may be processed from apolycistronic RNA after transcription of the polycistronic transcriptionunit by use of at least one selected from the group consisting of: anRNA cleavage site, a Csy4 cleavage site, a ribozyme cleavage site, apolynucleotide guided polypeptide cleavage site, the presence of a tRNAsequence, and any combination thereof. In some embodiments, thepolycistronic RNA may comprise a first tRNA sequence 5′ to the at leastone guide RNA and a second tRNA sequence 3′ to the at least one guideRNA.

In another embodiment, the method may comprise the eighthpolynucleotide, wherein the eighth polynucleotide may encode at leastone selected from the group consisting of: a herbicide toleranceprotein, a pesticidal protein, an accessory protein that binds to apesticidal protein, a dsRNA, a siRNA, a miRNA, and any combinationthereof, wherein the dsRNA, the siRNA and the miRNA suppress at leastone target gene present in a plant pest. In some embodiments, the methodmay comprise the eighth polynucleotide, wherein the eighthpolynucleotide may be operably linked to at least one regulatory elementthat is active in an organelle. In some embodiments, the at least oneregulatory element may be a promoter.

In another embodiment, at least one selected from the group consistingof: the first polynucleotide, the second polynucleotide, the fourthpolynucleotide, the fifth polynucleotide, and any combination thereof,may be located outside the region bounded by the sixth and the seventhpolynucleotide.

In another embodiment, the method may comprise the fourth and fifthpolynucleotides, wherein both the fourth and the fifth polynucleotidesmay be located outside the region bounded by the sixth and the seventhpolynucleotides.

In another embodiment, the method may comprise the fourthpolynucleotide, wherein the fourth polynucleotide may comprise a firstsequence encoding a positive selectable marker and a second sequenceencoding a negative selectable marker, wherein the first and the secondsequence may be each operably linked to a promoter that is functional inthe organelle.

In another embodiment, the method may comprise the fifth polynucleotide,wherein the fifth polynucleotide may encode an origin of replicationthat is functional in a plastid (e.g., a chloroplast), wherein theorigin of replication functional in a plastid may correspond to DNAsequence from a plastid rRNA intergenic region.

In another embodiment, the method may comprise the fifth polynucleotide,wherein the fifth polynucleotide may encode an origin of replicationthat is functional in a mitochondrion.

In some embodiments, the polynucleotide-guided polypeptide may beselected from the group consisting of: a Cas9 protein, a MAD2 protein, aMAD7 protein, a CRISPR nuclease, a nuclease domain of a Cas protein, aCpf1 protein, an Argonaute, modified versions thereof, and anycombination thereof.

In some embodiments, the recombinant DNA construct may further comprisea ninth and tenth polynucleotide that have at least 100 nucleotides of100 percent sequence identity to each other, wherein the ninth and tenthpolynucleotides are arranged as direct repeats in the recombinant DNAconstruct.

In some embodiments, the recombinant DNA construct may be linear andfurther wherein the ninth and tenth polynucleotides may be present atthe 5′ and 3′ ends of the recombinant DNA construct

In another embodiment, the method may comprise a recombinant DNAconstruct that comprises at least one selected from the group consistingof: the first polynucleotide, the second polynucleotide, the thirdpolynucleotide, the fourth polynucleotide, the fifth polynucleotide, andany combination thereof. In some embodiments, the method may comprisemore than one such recombinant DNA construct.

In another embodiment, the recombinant DNA construct may furthercomprise a ninth and tenth polynucleotide, wherein the ninth and tenthpolynucleotide may have 100 percent sequence identity to each other, andfurther wherein the ninth and tenth polynucleotides may be arranged asdirect repeats in the recombinant DNA construct. In some embodiments,the ninth and tenth polynucleotides may have at least 20, 21, 22, 23,24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides of 100 percentsequence identity to each other. Optionally, the recombinant DNAconstruct may be linear and the ninth and tenth polynucleotides arepresent at the 5′ and 3′ ends of the recombinant DNA construct.

In another embodiment, any of the methods herein may further involveintroducing into the organelle a polynucleotide encoding at least oneselectable marker selected from the group consisting of: a positiveselectable marker, a negative selectable marker, and any combinationthereof. In some embodiments, the positive selectable marker may be anherbicide tolerance protein. In some embodiments, the herbicidetolerance protein may be at least one selected from the group consistingof: a 4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide, an acetyl coenzyme A carboxylase(ACCase), and any combination thereof.

In some embodiments, the method may further involve growing the cell inthe presence of a positive selection agent and selecting a cell that ishomoplasmic for the altered genome of the organelle. In someembodiments, the method may further involve growing the cell in theabsence of the positive selection agent, followed by selecting a cellthat lacks a non-integrated recombinant DNA construct. In someembodiments, the method may further involve growing the cell in theabsence of the positive selection agent, followed by growing the cell inthe presence of a negative selection agent, followed by selecting a cellthat lacks a non-integrated recombinant DNA construct. In someembodiments, the cell may be selected from the group consisting of: ayeast cell, an algal cell, a plant cell, an insect cell, a non-humananimal cell, an isolated and purified human cell, and a mammalian tissueculture cell. In some embodiments, in the method for a plant cell, theorganelle may be a plastid (e.g., a chloroplast) or a mitochondrion. Insome embodiments, the method may further involve regenerating or growinga plant from the plant cell comprising an altered organelle genome. Insome embodiments, the plant cell may be monocot cell, e.g., a maizecell. The plant cell may be a dicot cell, e.g., a soybean cell.

In some embodiments, the cell maybe a plant cell, wherein the organelleis a plastid or a mitochondrion, and wherein the method furthercomprises regenerating a plant from the plant cell comprising an alteredorganelle genome. In some embodiments, the cell may be a yeast cell oran algal cell. In some embodiments, a plant, seed, root, stem, leaf,flower, fruit, or bean produced by the method disclosed herein maycomprise an organelle with an altered genome.

In another embodiment, the alteration of the genome of the organelle maycomprise an insertion of an expression cassette. In some embodiments,the expression cassette may be a polycistronic expression cassette. Insome embodiments, the polycistronic expression cassette may encode aselectable marker or a screenable marker, or both.

In another aspect, a recombinant DNA construct may comprise thefollowing: (i) a first polynucleotide encoding at least one guidepolynucleic acid, wherein the at least one guide polynucleic aciddirects a polynucleotide guided polypeptide to cleave at least onetarget sequence present in an organelle genome; (ii) a secondpolynucleotide encoding a polynucleotide guided polypeptide, wherein thepolynucleotide guided polypeptide, when associated with the guidepolynucleic acid, cleaves the at least one target sequence; (iii)optionally, a third polynucleotide encoding at least one homologousorganelle DNA sequence, wherein the at least one homologous organelleDNA is of sufficient size for homologous recombination, whereinintegration of the at least one homologous organelle DNA sequence intothe organelle genome results in removal of the at least one targetsequence; (iv) optionally, a fourth polynucleotide encoding at least oneselectable marker or at least one screenable marker, or both; whereinthe fourth polynucleotide is operably linked to a promoter that isfunctional in the organelle; and (v) optionally, a fifth polynucleotideencoding an origin of replication that is functional in the organelle.In some embodiments, the third polynucleotide of (iii) may comprise asixth and a seventh polynucleotide, wherein the sixth and the seventhpolynucleotides correspond to two adjacent regions of homology in theorganelle genome, wherein the sixth and seventh polynucleotides areseparated by a sequence that is heterologous to the organelle DNA. Insome embodiments, a yeast cell, algal cell, plant cell, plant, seed,root, stem, leaf, flower, fruit, or bean may comprise the recombinantDNA construct.

In another aspect, a recombinant DNA construct may comprise thefollowing: (i) a first polynucleotide encoding at least one guide RNA,wherein the at least one guide RNA directs a polynucleotide guidedpolypeptide to cleave at least one target sequence present in anorganelle genome; (ii) a second polynucleotide encoding a polynucleotideguided polypeptide, wherein the polynucleotide guided polypeptide, whenassociated with the guide RNA, cleaves the at least one target sequence;(iii) a third polynucleotide comprising a sixth and a seventhpolynucleotide, wherein the sixth and the seventh polynucleotidescorrespond to two adjacent regions of homology in the organelle genome,wherein the sixth and seventh polynucleotides are separated by asequence that is heterologous to the organelle DNA, wherein the sequencethat is heterologous to the organelle DNA comprises at least oneselected from the group consisting of: the first polynucleotide, thesecond polynucleotide, the fourth polynucleotide, an eighthpolynucleotide, and any combination thereof, wherein the eighthpolynucleotide encodes an RNA that is heterologous to the organelle;(iv) optionally, a fourth polynucleotide encoding at least oneselectable marker or at least one screenable marker, or both; whereinthe fourth polynucleotide is operably linked to a promoter that isfunctional in the organelle; and (v) optionally, a fifth polynucleotideencoding an origin of replication that is functional in the organelle.

In another aspect, a method for altering the genome of an organelle maycomprise: (a) introducing into a cell: (i) a polynucleotide encoding anRNA sequence comprising an organelle targeting RNA operably linked to aguide polynucleic acid, wherein the guide polynucleic acid directs apolynucleotide guided polypeptide to cleave a target sequence present inan organelle genome, wherein the polynucleotide is operably linked to atleast one regulatory element; and (ii) a second polynucleotide encodinga modified polynucleotide guided polypeptide, wherein the secondpolynucleotide is operably linked to at least one regulatory element,and wherein the modified polynucleotide guided polypeptide comprises apolynucleotide guided polypeptide operably linked to an organelletargeting peptide; wherein the organelle targeting RNA of (i) and theorganelle targeting peptide of (ii) each target the same organelle; and(b) growing the cell under conditions in which the polynucleotide of (i)and the second polynucleotide of (ii) are both expressed. In someembodiments, the method may further comprise a step (c) of selecting acell having an organelle that comprises an altered genome. In someembodiments, the method may further comprise a step (d) of selecting acell that is homoplasmic for the altered genome of the organelle.

In another aspect, a method for altering the genome of an organelle maycomprise: (a) introducing into a cell: (i) a polynucleotide encoding anRNA sequence comprising an organelle targeting RNA operably linked to aguide polynucleic acid, wherein the guide polynucleic acid directs apolynucleotide guided polypeptide to cleave a target sequence present inan organelle genome, wherein the polynucleotide is operably linked to atleast one regulatory element; and (ii) a third polynucleotide, whereinthe third polynucleotide is operably linked to at least one regulatoryelement, wherein the third polynucleotide encodes an RNA moleculecomprising an organelle targeting RNA operably linked to an RNA sequenceencoding a polynucleotide guided polypeptide; wherein the organelletargeting RNA of (i) and the organelle targeting RNA of (ii) each targetthe same organelle; and (b) growing the cell under conditions in whichthe polynucleotide of (i) and the third polynucleotide of (ii) are bothexpressed. In some embodiments, the method may further comprise a step(c) of selecting a cell having an organelle that comprises an alteredgenome. In some embodiments, the method may further comprise a step (d)of selecting a cell that is homoplasmic for the altered genome of theorganelle.

In another embodiment, any of the methods herein may further compriseintroducing a polynucleotide comprising at least one donorpolynucleotide (e.g. donor DNA) into the organelle, wherein the at leastone donor polynucleotide (e.g. donor DNA) is bounded by at least onehomologous sequence with respect to the organelle genome, whereinintegration of all or part of the at least one donor polynucleotide intothe organelle genome results in removal of the target site of the guidepolynucleic acid. In some embodiments, the at least one donorpolynucleotide (e.g. donor DNA) may comprise a first nucleic acidsequence heterologous to the organelle genome, wherein the first nucleicacid sequence is bounded by a second and a third nucleic acid sequence,wherein the second and the third nucleic acid sequences correspond totwo adjacent regions of homology in the organelle genome. In someembodiments, the second or the third nucleic acid sequence, or both, maycomprise at least one altered sequence, wherein the at least one alteredsequence is altered with respect to at least one additional target sitein the organelle genome, wherein the at least one altered sequence isnot recognized by at least one additional guide polynucleic acid,wherein the at least one additional guide polynucleic acid may direct apolynucleotide guided polypeptide to cleave the at least one additionaltarget site in the organelle genome. In some embodiments, the at leastone additional target site in the organelle genome may be present in atleast one essential coding region. In some embodiments, thepolynucleotide introduced into the organelle may further comprise afourth nucleic acid sequence, wherein the fourth nucleic acid sequenceencodes the at least one additional guide polynucleic acid. In someembodiments, the at least one additional guide polynucleic acid may beoperably linked to a promoter that is active in the organelle.

In some embodiments, the polynucleotide introduced into the organellefurther may comprise a fourth nucleic acid sequence, wherein the fourthnucleic acid sequence encodes the at least one additional guide RNAoperably linked to a promoter that is active in the organelle. In someembodiments, a cell produced by the method disclosed herein may beselected from the group consisting of: a yeast cell, an algal cell, aplant cell, an insect cell, a non-human animal cell, an isolated andpurified human cell, and a mammalian tissue culture cell. In someembodiments, a plant, seed, root, stem, leaf, flower, fruit, or beanproduced by the method disclosed herein may comprise an organelle withan altered genome.

In another aspect, a method for altering a genome of an organelle maycomprise: (a) introducing into an organelle of a cell the following: (i)at least one guide RNA, wherein the at least one guide RNA directs apolynucleotide guided polypeptide to cleave at least one target sequencepresent in the genome of the organelle; (ii) a polynucleotide guidedpolypeptide, wherein the polynucleotide guided polypeptide, whenassociated with the at least one guide RNA, cleaves the at least onetarget sequence; and (iii) a replacement DNA; and (b) selecting a cellcomprising an organelle comprising the replacement DNA. In someembodiments, the replacement DNA of step (a) part (iii) may comprisefragments of organellar DNA or a complete organellar DNA from acultivar, line, sub-species and other species and is distinct from thegenome of the organelle of step (a). In some embodiments, thereplacement DNA may be lacking the at least one target sequence. In someembodiments, after step (a) part (ii) and prior to step (a) part (iii),a cell may be selected in which the genome of the organelle has beeneliminated. In some embodiments, the at least one target sequence maynot be present in the replacement DNA.

In some embodiments, the guide polynucleic acid in the methods andcompositions of matter described herein may comprise the following: i)at least 17 nucleotides that are complementary to at least 17nucleotides of a target polynucleic acid, wherein said targetpolynucleic acid is located in the genome of an organelle; and ii) aregion that contacts a polynucleotide-guided polypeptide. The guidepolynucleic acid may comprise one or more RNA bases. In someembodiments, the guide polynucleic acid may be a guide RNA. The guidepolynucleic acid may be a dual guide RNA. In some embodiments, the guidepolynucleic acid may be a single guide RNA.

In another embodiment, the polynucleotide-guided polypeptide in themethods and compositions of matter described herein may be selected fromthe group consisting of: a Cas9 protein, a MAD2 protein, a MAD7 protein,a CRISPR nuclease, a nuclease domain of a Cas protein, a Cpf1 protein,an Argonaute, modified versions thereof, and any combination thereof. Insome embodiments, the sequence encoding the polynucleotide-guidedpolypeptide may be codon-optimized for a human, a yeast, an alga, or aplant species.

In another embodiment, the cell may be a plant cell, the organelle maybe a plastid (e.g., a chloroplast) or a mitochondrion, and the methodmay further comprise regenerating or growing a plant from the plant cellcomprising an altered organelle genome.

In another embodiment, a cell produced by any of the methods describedherein may be selected from the group consisting of: a yeast cell, analgal cell, a plant cell, an insect cell, a non-human animal cell, anisolated and purified human cell, and a mammalian tissue culture cell.

In another embodiment, a plant, seed, root, stem, leaf, flower, fruit,or bean produced by any of the methods described herein may comprise anorganelle with an altered genome.

In another embodiment, a cell comprising any of the recombinant DNAconstructs described herein may be selected from the group consistingof: a yeast cell, an algal cell, a plant cell, an insect cell, anon-human animal cell, an isolated and purified human cell, and amammalian tissue culture cell.

In another embodiment, a plant, seed, root, stem, leaf, flower, fruit,or bean comprising any of the recombinant DNA constructs describedherein may comprise an organelle with an altered genome.

In one embodiment, a polynucleotide may comprise a) an organelletargeting sequence; and b) a guide polynucleic acid, wherein the guidepolynucleic acid comprises i) at least 17 nucleotides that arecomplementary to at least 17 nucleotides of a target polynucleic acid,wherein said target polynucleic acid is located in the genome of anorganelle; and ii) a region that contacts a polynucleotide-guidedpolypeptide, wherein said organelle targeting sequence and said guidepolynucleic acid sequence are operably linked. In another embodiment,the polynucleotide comprises one or more RNA bases. In anotherembodiment, the polynucleotide further comprises a sequence encoding thepolynucleotide-guided polypeptide. In another embodiment, saidpolynucleotide-guided polypeptide is a Cas9 protein. In anotherembodiment, said polynucleotide-guided polypeptide is an Argonauteprotein. In another embodiment, said polynucleotide-guided polypeptideis a nuclease in a CRISPR family. In another embodiment, saidpolynucleotide-guided polypeptide is Cpf1. In another embodiment, thesequence encoding said polynucleotide-guided polypeptide iscodon-optimized for a human. In another embodiment, the sequenceencoding said polynucleotide-guided polypeptide is codon-optimized for aplant species. In another embodiment, said target polynucleic acidcomprises a protospacer adjacent motif (PAM) sequence. In anotherembodiment, said Cas9 has been engineered to associate with an alteredPAM sequence. In another embodiment, said polynucleotide-guidedpolypeptide selectively cleaves the target polynucleic acid. In anotherembodiment, said polynucleotide-guided polypeptide selectively induces adouble-strand break in the target polynucleic acid. In anotherembodiment, said polynucleotide-guided polypeptide comprises a nucleasedomain that induces a nick in the target polynucleic acid. In anotherembodiment, the polynucleotide comprises two or more different guidepolynucleic acids. In another embodiment, the guide polynucleic acid iscomprised of a dual-guide RNA. In another embodiment, the guidepolynucleic acid is a single guide RNA. In another embodiment, the guidepolynucleic acid is comprised of a crRNA and a trRNA, wherein said crRNAand said trRNA are optionally linked. In another embodiment, said guidepolynucleic acid comprises a region that is engineered to becomplementary to at least 18 nucleotides of the target polynucleic acidin the organelle of a cell. In another embodiment, said guidepolynucleic acid is engineered to be substantially complementary to atleast 22 nucleic acids of the target polynucleic acid in the organelleof a cell. In another embodiment, said at least 17 nucleotides arecontiguous. In another embodiment, said organelle is a mitochondrion. Inanother embodiment, said organelle is a plastid. In another embodiment,said guide polynucleic acid is engineered to hybridize to a region of atarget gene disclosed herein. In another embodiment, the polynucleotidefurther comprises a modified RNA donor sequence, wherein the modifiedRNA donor sequence comprises an organelle targeting RNA operably linkedto a donor RNA.

In another embodiment. a DNA sequence that when translated to RNA mayresult in a polynucleotide of the disclosure.

In another embodiment, a polynucleotide encoding an RNA sequence maycomprise an organelle targeting RNA operably linked to a guide RNA,wherein the guide RNA directs a polynucleotide guided polypeptide tocleave a target sequence present in an organelle genome. The RNAsequence may further comprise a sequence encoding a polynucleotideguided polypeptide, and optionally, an RNA cleavage site between theguide RNA and the sequence encoding a polynucleotide guided polypeptide.

In another embodiment, an organelle may comprise the polynucleotide ofthe disclosure. In some embodiments, the organelle is a mitochondrion.In some embodiments, the organelle is a plastid.

In another embodiment, a cell may comprise any of the polynucleotides ofthe disclosure. The cell may further comprise a polynucleotide encodinga modified polynucleotide guided polypeptide, wherein the modifiedpolynucleotide guided polypeptide comprises a polynucleotide guidedpolypeptide operably linked to an organelle targeting peptide.

In another embodiment, a method for introducing a guide polynucleic acidinto an organelle of a cell may comprise: (a) introducing into a cell apolynucleotide encoding an RNA sequence comprising an organelletargeting RNA operably linked to a guide polynucleic acid, wherein theguide polynucleic acid directs a polynucleotide guided polypeptide tocleave a target sequence present in an organelle genome, further whereinthe polynucleotide is operably linked to at least one regulatoryelement; and (b) growing the cell under conditions in which thepolynucleotide is expressed.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell: (i) a polynucleotide encodingan RNA sequence comprising an organelle targeting RNA operably linked toa guide polynucleic acid, wherein the guide polynucleic acid directs apolynucleotide guided polypeptide to cleave a target sequence present inan organelle genome, wherein the polynucleotide is operably linked to atleast one regulatory element; and (ii) a second polynucleotide encodinga modified polynucleotide guided polypeptide, wherein the secondpolynucleotide is operably linked to at least one regulatory element,and wherein the modified polynucleotide guided polypeptide comprises apolynucleotide guided polypeptide operably linked to an organelletargeting peptide; wherein the organelle targeting RNA of (i) and theorganelle targeting peptide of (ii) each target the same organelle; and(b) growing the cell under conditions in which the polynucleotide of (i)and the second polynucleotide of (ii) are both expressed.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell: (i) a polynucleotide encodingan RNA sequence comprising an organelle targeting RNA operably linked toa guide polynucleic acid, wherein the guide polynucleic acid directs apolynucleotide guided polypeptide to cleave a target sequence present inan organelle genome, wherein the polynucleotide is operably linked to atleast one regulatory element; and (ii) a third polynucleotide, whereinthe third polynucleotide is operably linked to at least one regulatoryelement, wherein the third polynucleotide encodes an RNA moleculecomprising an organelle targeting RNA operably linked to an RNA sequenceencoding a polynucleotide guided polypeptide; wherein the organelletargeting RNA of (i) and the organelle targeting RNA of (ii) each targetthe same organelle; and (b) growing the cell under conditions in whichthe polynucleotide of (i) and the third polynucleotide of (ii) are bothexpressed.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell a polynucleotide encoding anRNA sequence comprising: (i) an organelle targeting RNA operably linkedto a guide polynucleic acid, wherein the guide polynucleic acid isdirects a polynucleotide guided polypeptide to cleave a target sequencepresent in an organelle genome, (ii) a sequence encoding apolynucleotide guided polypeptide, and (iii) an RNA cleavage sitebetween the guide polynucleic acid and the sequence encoding apolynucleotide guided polypeptide, wherein the polynucleotide isoperably linked to at least one regulatory element; and (b) growing thecell under conditions in which the polynucleotide of (a) is expressed.

In another embodiment, any of the methods herein may further compriseintroducing a polynucleotide comprising at least one donorpolynucleotide (e.g. donor DNA) into the organelle, wherein the at leastone donor polynucleotide (e.g. donor DNA) is bounded by at least onehomologous sequence with respect to the organelle genome, whereinintegration of all or part of the at least one donor polynucleotide intothe organelle genome results in removal of the target site of the guidepolynucleic acid. The at least one donor polynucleotide (e.g. donor DNA)may comprise a first nucleic acid sequence heterologous to the organellegenome, wherein the first nucleic acid sequence is bounded by a secondand a third nucleic acid sequence, wherein the second and the thirdnucleic acid sequences correspond to two adjacent regions of homology inthe organelle genome. Additionally, the second or the third nucleic acidsequence, or both, may comprise at least one altered sequence, whereinthe at least one altered sequence is altered with respect to at leastone additional target site in the organelle genome, wherein the at leastone altered sequence is not recognized by at least one additional guidepolynucleic acid, wherein the at least one additional guide polynucleicacid directs a polynucleotide guided polypeptide to cleave the at leastone additional target site in the organelle genome. The at least oneadditional target site in the organelle genome may be present in atleast one essential coding region. The polynucleotide introduced intothe organelle may further comprise a fourth nucleic acid sequence,wherein the fourth nucleic acid sequence encodes the at least oneadditional guide polynucleic acid operably linked to a promoter that isactive in the organelle.

In another embodiment, a polynucleotide may encode a modified RNA donorsequence, wherein the modified RNA donor sequence may comprise anorganelle targeting RNA operably linked to a donor RNA. The modified RNAdonor sequence may comprise a reverse transcriptase primer site.Additionally, a cell comprising the polynucleotide, and furthercomprising a polynucleotide encoding a modified reverse transcriptase,wherein the modified reverse transcriptase comprises a reversetranscriptase operably linked to an organelle targeting peptide.

In another embodiment, a method of altering the genome of an organellemay further comprise introducing a donor polynucleotide into theorganelle, wherein the donor polynucleotide is introduced into theorganelle by: (a) introducing the polynucleotide encoding a modified RNAdonor sequence into the cell, wherein the polynucleotide is operablylinked to at least one regulatory element; (b) introducing into the cella polynucleotide encoding a modified reverse transcriptase, wherein themodified reverse transcriptase comprises a reverse transcriptaseoperably linked to an organelle targeting peptide, wherein thepolynucleotide is operably linked to at least one regulatory element,wherein the organelle targeting RNA of (a) and the organelle targetingpeptide of (b) each target the same organelle; and (c) growing the cellunder conditions wherein the polynucleotides of (a) and (b) are bothexpressed.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into an organelle a recombinant DNAconstruct comprising the following: (i) a first polynucleotide encodingat least one guide polynucleic acid, wherein the at least one guidepolynucleic acid directs a polynucleotide guided polypeptide to cleaveat least one target sequence present in an organelle genome; (ii) asecond polynucleotide encoding a polynucleotide guided polypeptide,wherein the polynucleotide guided polypeptide, when associated with theguide polynucleic acid, cleaves the at least one target sequence; (iii)a third polynucleotide encoding at least one homologous organelle DNAsequence, wherein the at least one homologous organelle DNA is ofsufficient size for homologous recombination, wherein integration of theat least one homologous organelle DNA sequence into the organelle genomeresults in removal of the at least one target sequence; (iv) optionally,a fourth polynucleotide encoding at least one selectable marker; whereinthe fourth polynucleotide is operably linked to a promoter that isfunctional in the organelle; and (v) optionally, a fifth polynucleotideencoding an origin of replication that is functional in the organelle;and (b) growing a cell comprising the organelle of (a) under conditionsin which the first polynucleotide of (i) and the second polynucleotideof (ii) are each expressed. The third polynucleotide of (iii) maycomprise a sixth and a seventh polynucleotide, wherein the sixth and theseventh polynucleotides correspond to two adjacent regions of homologyin the organelle genome, wherein the sixth and seventh polynucleotidesare separated by a sequence that is heterologous to the organelle DNA,wherein the sequence that is heterologous to the organelle DNA comprisesat least one selected from the group consisting of: the firstpolynucleotide, the second polynucleotide, the fourth polynucleotide andan eighth polynucleotide, wherein the eighth polynucleotide encodes anRNA that is heterologous to the organelle.

In another embodiment, a method wherein at least one selected from thegroup consisting of: the first polynucleotide, the secondpolynucleotide, the fourth polynucleotide and the fifth polynucleotide,may be located outside the region bounded by the sixth and the seventhpolynucleotide.

In another embodiment, a method wherein both the fourth and the fifthpolynucleotides may be located outside the region bounded by the sixthand the seventh polynucleotides.

In another embodiment, the fourth polynucleotide comprises a firstsequence encoding a positive selectable marker and a second sequenceencoding a negative selectable marker, wherein the first and the secondsequence are each operably linked to a promoter that is functional inthe organelle.

In another embodiment, the fifth polynucleotide encodes a plastid originof replication, wherein the plastid origin of replication corresponds toDNA sequence from a plastid rRNA intergenic region.

In another embodiment, the fifth polynucleotide encodes a mitochondrialorigin of replication.

In another embodiment, the recombinant DNA construct further comprisesan eighth and ninth polynucleotide, wherein the eighth and ninthpolynucleotide have at least 100 nucleotides of 100 percent sequenceidentity to each other, wherein the eighth and ninth polynucleotides arearranged as direct repeats in the recombinant DNA construct. Optionally,the recombinant DNA construct is linear and the eighth and ninthpolynucleotides are present at the 5′ and 3′ ends of the recombinant DNAconstruct.

In another embodiment, the recombinant DNA construct is linear andsingle-stranded, and the recombinant DNA construct is operably linked toa modified VirD2 protein, wherein the modified VirD2 protein comprises aVirD2 protein operably linked to an organelle targeting peptide, whereinthe modified VirD2 protein has also been modified such that each nativenuclear localization sequence of the VirD2 protein is no longerfunctional. Optionally, the recombinant DNA construct is operably linkedto at least one modified VirE2 protein, wherein the at least onemodified VirE2 protein comprises a VirE2 protein operably linked to anorganelle targeting peptide, wherein the at least one modified VirE2protein has also been modified such that each native nuclearlocalization sequence of the VirE2 protein is no longer functional.Optionally, the recombinant DNA construct is operably linked to at leastone modified RecA protein, wherein the at least one modified RecAprotein comprises a RecA protein operably linked to an organelletargeting peptide. Optionally, the recombinant DNA construct is operablylinked to at least one chimeric polypeptide, wherein the at least onechimeric polypeptide comprises an organelle targeting peptide and a cellpenetrating peptide.

In another embodiment, any of the methods herein may further involveintroducing into the organelle a polynucleotide encoding at least oneselectable marker selected from the group consisting of: a positiveselectable marker, a negative selectable marker, and any combinationthereof. The positive selectable marker may be an herbicide toleranceprotein. The herbicide tolerance protein may be at least one selectedfrom the group consisting of: a 4-hydroxphenylpyruvate dioxygenase(HPPD), a sulfonylurea-tolerant acetolactate synthase (ALS), animidazolinone-tolerant acetolactate synthase (ALS), aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),a glyphosate-tolerant glyphosate oxidoreductase (GOX), a glyphosateN-acetyltransferase (GAT), a phosphinothricin acetyl transferase (PAT),a protoporphyrinogen oxidase (PROTOX), an auxin enzyme or receptor, aP450 polypeptide and an acetyl coenzyme A carboxylase (ACCase). Themethod may further involve growing the cell in the presence of apositive selection agent and selecting a cell that is homoplasmic forthe altered genome of the organelle. Optionally, the method may furtherinvolve growing the cell in the absence of the positive selection agent,followed by selecting a cell that lacks a non-integrated recombinant DNAconstruct. Alternatively, the method may further involve growing thecell in the absence of the positive selection agent, followed by growingthe cell in the presence of a negative selection agent, followed byselecting a cell that lacks a non-integrated recombinant DNA construct.In the method, the cell may be a plant cell, the organelle may be aplastid. The method may further involve regenerating a plant from theplant cell comprising an altered organelle genome. The plant cell may bemonocot cell, e.g., a maize cell. The plant cell may be a dicot cell,e.g., a soybean cell.

In another embodiment, in any of the methods herein for altering thegenome of an organelle to contain a heterologous polynucleotide, theheterologous polynucleotide may encode at least one selected from thegroup consisting of: a herbicide tolerance protein, a pesticidalprotein, an accessory protein that binds to a pesticidal protein, adsRNA, a siRNA and a miRNA, wherein the dsRNA, the siRNA and the miRNAsuppress at least one target gene present in a plant pest. The herbicidetolerance protein may be at least one selected from the group consistingof: a 4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide and an acetyl coenzyme Acarboxylase (ACCase). The pesticidal protein may be at least oneselected from the group consisting of: Cry1Ac, Cyt1Aa, Cry1Ab, Cry2Aa,Cry1I, Cry1C, Cry1D, Cry1E, Cry1Be, Cry1Fa and Vip3A. The accessoryprotein that binds to a pesticidal protein may be at least one selectedfrom the group consisting of: a 20 kDa accessory protein and a 19 kDaaccessory protein. The dsRNA, the siRNA and the miRNA can suppress atleast one target gene selected from the group consisting of: proteasomeA-type subunit peptide (Pas-4), ACT, SHR, EPIC2B and PnPMAI. Theheterologous polynucleotide may be operably linked to at least oneregulatory element that is active in an organelle. The at least oneregulatory element may be selected from the group consisting of: a maizeclpP promoter combined with a maize clpP 5′-UTR, a maize clpP promotercombined with a 5′-UTR from gene 10 of bacteriophage T7, a tomato psbApromoter is combined with a 5′-UTR from gene 10 of bacteriophage T7 anda tomato rm16 promoter combined with a modified accD 5′-UTR. The cellmay be a plant cell, wherein the organelle is a plastid, and wherein themethod further comprises regenerating a plant from the plant cellcomprising an altered organelle genome. The plant cell may be a soybeancell.

In another embodiment, a cell may comprise an organelle with an alteredgenome, wherein the cell may be produced by any of the above methods.The cell may be selected from the group consisting of: a yeast cell, analgal cell, a plant cell, an insect cell, a non-human animal cell, anisolated and purified human cell, and a mammalian tissue culture cell.

In another embodiment, a method may comprise altering the genome of anorganelle in a cell as described above, wherein the cell is a plant celland further wherein a plant is regenerated from a plant cell, whereinthe plant comprises an organelle with an altered genome. Also, a plant(e.g., progeny plant) or seed produced from the regenerated plant,wherein the plant or seed comprises an organelle with an altered genome.

In another embodiment, a plant, seed, root, stem, leaf, flower, fruit,or bean may be produced by a method of the disclosure. In someembodiments, the plant, seed, root, stem, leaf, flower, fruit, or beancomprises an organelle with an altered genome.

In another embodiment, a plant, seed, root, stem, leaf, flower, fruit,or bean may comprise a polynucleotide of the disclosure.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1 presents the sequence (SEQ ID NO: 144) obtained from PCRamplification of the replaced DNA locus in transformed yeastmitochondrial DNA modified by the Edit Plasmid approach; and

FIG. 2 presents the sequence (SEQ ID NO: 171) obtained from PCRamplification of the replaced DNA locus in transformed Chlamydomonasplastid DNA modified by the Edit Plasmid approach.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The disclosure is more fully understood from the following detaileddescription and Sequence Listing, which form a part of this application.

SEQ ID NO: 1 corresponds to the nucleic acid sequence encoding mCas9-A;i.e., a Cas9 comprising ATPase beta mitochondrial targeting peptide.

SEQ ID NO: 2 corresponds to the nucleic acid sequence encoding mCas9-B;i.e., a Cas9 comprising the 70 kD mitochondrial targeting peptide.

SEQ ID NO: 3 corresponds to the nucleic acid sequence encoding a guideRNA-tRNA^(Lys) (tRK1) fusion (“N” residues indicate the variabletargeting domain of the guide RNA).

SEQ ID NO: 4 corresponds to the nucleic acid sequence encoding a guideRNA-tRNA^(Lys) fusion (tRK2-2 version for mitochondrial import; “N”residues indicate the variable targeting domain of the guide RNA).

SEQ ID NO: 5 corresponding to the nucleic acid sequence encoding a guideRNA-tRNA^(Lys) fusion with an altered 5′ tRNA end.

SEQ ID NO: 6 corresponds to the nucleic acid sequence encoding a guideRNA-tRNA^(Lys) fusion (modified tRK2 version with altered 5′ end; “N”residues at the 5′ end indicate the variable targeting domain of theguide RNA).

SEQ ID NO: 7 corresponds to the nucleic acid sequence encoding a gRNAembedded in tRK2 intron in the backbone of tRK2-2 (20-mer of “N”residues indicates the variable targeting domain; 3-mer of “N” residuesis complementary to the first three nucleotides of the variabletargeting domain to preserve the secondary structure for splicing).

SEQ ID NO: 8 corresponds to the nucleic acid sequence encoding a gRNAembedded in tRK2 type intron in the backbone of tRK1 (20-mer of “N”residues indicates the variable targeting domain; 3-mer of “N” residuesis complementary to the first three nucleotides of guide RNA to preservethe secondary structure for splicing).

SEQ ID NO: 9 corresponds to the nucleic acid sequence encoding a gRNAfused with second half of tRK1 (B form).

SEQ ID NO: 10 corresponds to the nucleic acid sequence encoding a formof tRK1 to be co-expressed with guide RNA-B form fusion.

SEQ ID NO: 11 corresponds to the nucleic acid sequence encoding a gRNAconstructed between the D arm and F hairpin structures.

SEQ ID NO: 12 corresponds to the nucleic acid sequence encoding a gRNAfused with the D arm.

SEQ ID NO: 13 corresponds to the nucleic acid sequence encoding a gRNAfused with F hairpin structure.

SEQ ID NO: 14 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the cytochrome b gene inmitochondria.

SEQ ID NO: 15 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the COX1 gene in mitochondria.

SEQ ID NO: 16 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the COX1 gene in mitochondria.

SEQ ID NO: 17 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the COX2 gene in mitochondria.

SEQ ID NO: 18 corresponds to the nucleic acid sequence that is fusedwith the 3′ end of a variable targeting domain to create a functionalguide RNA for Cas9.

SEQ ID NO: 19 corresponds to the nucleic acid sequence encoding a SNR52promoter.

SEQ ID NO: 20 corresponds to the nucleic acid sequence encoding a SUP4Terminator.

SEQ ID NO: 21 corresponds to the nucleic acid sequence for aoligonucleotide primer for paromomycin-resistance template DNA

SEQ ID NO: 22 corresponds to the nucleic acid sequence for acomplementary oligonucleotide primer to make template DNA with theprimer of SEQ ID NO: 21.

SEQ ID NO: 23 corresponds to the nucleic acid sequence encoding thevariable targeting domain for a guide RNA that targets the 15S rRNA genein mitochondria.

SEQ ID NO: 24 corresponds to a nucleic acid sequence encoding a Cas9gene optimized for expression in yeast mitochondria.

SEQ ID NO: 25 corresponds to the nucleic acid sequence encoding a COX2promoter.

SEQ ID NO: 26 corresponds to the nucleic acid sequence encoding a COX2terminator.

SEQ ID NO: 27 corresponds to the nucleotide sequence of the variabletargeting domain for a guide RNA to target the mitochondrial 21S rRNAgene in yeast.

SEQ ID NO: 28 corresponds to the nucleic acid sequence encoding thepromoter sequence of the 15S rRNA gene.

SEQ ID NO: 29 corresponds to the nucleic acid sequence encoding theterminator sequence of the 15S rRNA gene.

SEQ ID NO: 30 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the COB gene in mitochondria.

SEQ ID NO: 31 corresponds to the nucleotide sequence for the variabletargeting domain of a guide RNA to target the ATP9 gene in mitochondria.

SEQ ID NO: 32 corresponds to the amino acid sequence for the NDUFV2mitochondrial targeting peptide.

SEQ ID NO: 33 corresponds to the nucleic acid sequence encoding a Cas9fused with a mitochondrial targeting peptide derived from NDUFV2.

SEQ ID NO: 34 corresponds to the amino acid sequence of themitochondrial targeting peptide of citrate synthase.

SEQ ID NO: 35 corresponds to the nucleic acid sequence encoding a Cas9fused with the mitochondrial signal peptide derived from human citratesynthase.

SEQ ID NO: 36 corresponds to the nucleic acid sequence encoding a human5S rRNA gene for mitochondrial import (the 4-mer “GTCT can be replacedwith guide RNA).

SEQ ID NO: 37 corresponds to the nucleotide sequence of a variabletargeting domain for a gRNA sequence targeting the human COX3 gene inmitochondria.

SEQ ID NO: 38 corresponds to the nucleic acid sequence of an expressioncassette for a guide RNA utilizing the promoter and terminator of thehuman 5S rRNA gene.

SEQ ID NO: 39 corresponds to the nucleotide sequence of a variabletargeting domain for a guide RNA to target the CAPR locus in mousemitochondrial DNA (CAP^(R) allele has an A to G substitution at residue17).

SEQ ID NO: 40 corresponds to the nucleotide sequence of a polynucleotidemodification template with the CAP^(R) mutation (part of the mouse16SrRNA).

SEQ ID NO: 41 corresponds to the nucleotide sequence encoding pcoCas9without NLS & FLAG domains, but with the potato IV intron. The sequenceis codon-optimized for Arabidopsis (GenBank ID: KF264451).

SEQ ID NO: 42 corresponds to the amino acid sequence of pcoCas9.

SEQ ID NO: 43 corresponds to the amino acid sequence of the transitpeptide of AtRbcS (At1g67090). Cleavage occurs after the “N” residue atposition 54.

SEQ ID NO: 44 corresponds to the amino acid sequence of the transitpeptide of AtCab (NP_001078288.1). Cleavage occurs after the “P” residueat position 55.

SEQ ID NO: 45 corresponds to the amino acid sequence of the transitpeptide of At DnaJ8 (NP_178207.1). Cleavage occurs after the “V” residueat position 47.

SEQ ID NO: 46 corresponds to the nucleotide sequence encoding thepcoCas9 with AT-rbcS transit peptide (with potato intron).

SEQ ID NO: 47 corresponds to the amino acid sequence of pcoCas9 withAT-rbcS chloroplast transit peptide.

SEQ ID NO: 48 corresponds to the nucleotide sequence encoding the Vd5′UTR (gi|301016157|gb|HM136583.1|.

SEQ ID NO: 49 corresponds to the nucleotide sequence encoding theAteIF4E1 full-length cDNA.

SEQ ID NO: 50 corresponds to the nucleotide sequence encoding a typicalgRNA module (5′ terminal 20-mer of “N” residues corresponds to thevariable targeting domain).

SEQ ID NO: 51 corresponds to the nucleotide sequence encoding CSY4.

SEQ ID NO: 52 corresponds to the amino acid sequence of the Csy4polypeptide.

SEQ ID NO: 53 corresponds to the nucleotide sequence of the Csy4recognition site.

SEQ ID NO: 54 corresponds to the nucleotide sequence encoding a guideRNA flanked by Csy4 recognition sites (multimeric form).

SEQ ID NO: 55 corresponds to the nucleotide sequence encoding aNt_Chl_rpoB (Nicotiana tabacum RNA polymerase beta chain).

SEQ ID NO: 56 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rpoB gene from Nicotianatabacum.

SEQ ID NO: 57 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rpoB gene from Nicotianatabacum.

SEQ ID NO: 58 corresponds to the nucleotide sequence encoding aNt_Cp_psbA (Nicotiana tabacum photosystem II protein D1).

SEQ ID NO: 59 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid psbA gene from Nicotianatabacum.

SEQ ID NO: 60 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid psbA gene from Nicotianatabacum.

SEQ ID NO: 61 corresponds to the nucleotide sequence encoding aNt_Cp_rps15 (Nicotiana tabacum ribosomal protein S15).

SEQ ID NO: 62 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps15 gene from Nicotianatabacum.

SEQ ID NO: 63 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps15 gene from Nicotianatabacum.

SEQ ID NO: 64 corresponds to the nucleotide sequence encoding aNt_Cp_rp133 (Nicotiana tabacum 50S ribosomal protein L33).

SEQ ID NO: 65 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rp133 gene from Nicotianatabacum.

SEQ ID NO: 66 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rp133 gene from Nicotianatabacum.

SEQ ID NO: 67 corresponds to the nucleotide sequence encoding a GlmaCprpoB (Glycine max RNA polymerase beta chain).

SEQ ID NO: 68 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rpoB gene from Glycine max.

SEQ ID NO: 69 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rpoB gene from Glycine max.

SEQ ID NO: 70 corresponds to the nucleotide sequence encoding a GlmaCppsbA (Glycine max photosystem II protein D1).

SEQ ID NO: 71 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid psbA gene from Glycine max.

SEQ ID NO: 72 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid psbA gene from Glycine max.

SEQ ID NO: 73 corresponds to the nucleotide sequence encoding aGlmaCp_rps15 (Glycine max ribosomal protein S15).

SEQ ID NO: 74 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps15 gene from Glycinemax.

SEQ ID NO: 75 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps15 gene from Glycinemax.

SEQ ID NO: 76 corresponds to the nucleotide sequence encoding aGlmaCp_rp133 (Glycine max 505 ribosomal protein L33).

SEQ ID NO: 77 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rp133 gene from Glycinemax.

SEQ ID NO: 78 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rp133 gene from Glycinemax.

SEQ ID NO: 79 corresponds to the nucleotide sequence encoding aNicotiana benthamiana rps16 with intron (ribosomal protein S16, GI:KC495035.1).

SEQ ID NO: 80 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps16 gene from Nicotianabenthamiana.

SEQ ID NO: 81 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid rps16 gene from Nicotianabenthamiana.

SEQ ID NO: 82 corresponds to the nucleotide sequence encoding aNicotiana benthamiana matK (maturase K, GI: AB040014).

SEQ ID NO: 83 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid matK gene from Nicotianabenthamiana.

SEQ ID NO: 84 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting the plastid matK gene from Nicotianabenthamiana.

SEQ ID NO: 85 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region (NtChrC; 57408 . .. 57389) from Nicotiana tabacum.

SEQ ID NO: 86 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region (NtChrC; 59412 . .. 59393) from Nicotiana tabacum.

SEQ ID NO: 87 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region (NtChrC; 59622 . .. 59603) from Nicotiana tabacum.

SEQ ID NO: 88 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region (NtChrC; 65704 . .. 65723) from Nicotiana tabacum.

SEQ ID NO: 89 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region(GlmaCp_NC_007942.1_59039-59058) from Glycine max.

SEQ ID NO: 90 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region(GlmaCp_NC_007942.1_59100-59119) from Glycine max.

SEQ ID NO: 91 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region(GlmaCp_NC_007942.1_62057-62038) from Glycine max.

SEQ ID NO: 92 corresponds to the nucleotide sequence of variable targetregion for a guide RNA targeting an intergenic region(GlmaCp_NC_007942.1_62361-62380) from Glycine max.

SEQ ID NO: 93 corresponds to the nucleotide sequence of the target sitefor the plastid psbA gene.

SEQ ID NO: 94 corresponds to the nucleotide sequence of the region ofthe polynucleotide modification template that corresponds to the targetsite of the plastid psbA gene

SEQ ID NO: 95 corresponds to the amino acid sequence of the ATPase Betamitochondrial targeting peptide, which is encoded by SEQ ID NO:1.

SEQ ID NO: 96 corresponds to the amino acid sequence of the Cas9polypeptide fused to the ATPase Beta mitochondrial targeting peptide,which is encoded by SEQ ID NO:1.

SEQ ID NO: 97 corresponds to the amino acid sequence of the 70 kDmitochondrial targeting peptide, which is encoded by SEQ ID NO:2.

SEQ ID NO: 98 corresponds to the amino acid sequence of the Cas9polypeptide fused to the 70 kD mitochondrial targeting peptide, which isencoded by SEQ ID NO:2.

SEQ ID NO: 99 corresponds to the nucleotide sequence of the forwardprimer ZmPclpP-Forward, for PCR amplification of the maize clpP promoterin combination with the clpP 5′-UTR (ZmPclpP:clpP). This forward primermay also be used for PCR amplification of the maize clpP promoter incombination with the 5′-UTR from gene 10 of bacteriophage T7(ZmPclpP:G10).

SEQ ID NO: 100 corresponds to the nucleotide sequence of the reverseprimer ZmPclpP-Reverse, for PCR amplification of the maize clpP promoterin combination with the clpP 5′-UTR (ZmPclpP:clpP).

SEQ ID NO: 101 corresponds to the nucleotide sequence of the reverseprimer for PCR amplification of the maize clpP promoter in combinationwith the 5′-UTR from gene 10 of bacteriophage T7 (ZmPclpP:G10).

SEQ ID NO: 102 corresponds to the nucleotide sequence of the forwardprimer for PCR amplification of the tomato psbA promoter in combinationwith the 5′-UTR from gene 10 of bacteriophage T7 (SlPsbA:T7g10).

SEQ ID NO: 103 corresponds to the nucleotide sequence of the reverseprimer for PCR amplification of the tomato psbA promoter in combinationwith the 5′-UTR from gene 10 of bacteriophage T7 (SlPsbA:T7g10).

SEQ ID NO: 104 corresponds to the nucleotide sequence of the forwardprimer for PCR amplification of the SIPrm16 promoter portion of thetomato rm16 promoter in combination with the accD-mod 5′-UTR.

SEQ ID NO: 105 corresponds to the nucleotide sequence of the reverseprimer for PCR amplification of the SIPrm16 promoter portion of thetomato rm16 promoter in combination with the accD-mod 5′-UTR.

SEQ ID NO: 106 corresponds to the nucleotide sequence of the forwardprimer for PCR amplification of the accD-mod 5′-UTR portion of thetomato rm16 promoter in combination with the accD-mod 5′-UTR.

SEQ ID NO: 107 corresponds to the nucleotide sequence of the reverseprimer for PCR amplification of the accD-mod 5′-UTR portion of thetomato rm16 promoter in combination with the accD-mod 5′-UTR.

SEQ ID NO: 108 corresponds to the nucleotide sequence from Bacillusthuringiensis serovar kurstaki HD73 that encodes a Cry1Acdelta-endotoxin (U89872).

SEQ ID NO: 109 corresponds to the amino acid sequence of the Cry1Acdelta-endotoxin encoded by SEQ ID NO: 108.

SEQ ID NO: 110 corresponds to the nucleotide sequence from Bacillusthuringiensis serovar kurstaki HD73 that encodes a truncated form of aCry1Ac delta-endotoxin that has insecticidal activity.

SEQ ID NO: 111 corresponds to the nucleotide sequence from Bacillusthuringiensis serovar israelensis that encodes a Cyt1Aa protein (GeneID: 5759908).

SEQ ID NO: 112 corresponds to the nucleotide sequence from Bacillusthuringiensis serovar israelensis (pBt024) that encodes a 20 kDaaccessory protein.

SEQ ID NO: 113 corresponds to the nucleotide sequence from Bacillusthuringiensis serovar israelensis (pBt022) that encodes a 19 kDaaccessory protein.

SEQ ID NO: 114 corresponds to the nucleotide sequence for an openreading frame encoding an Heterodera glycines (SCN) specific proteasomeA-type subunit peptide referred to herein as Pas-4 (U58067671).

SEQ ID NO: 115 corresponds to nucleotides 552-699 of SEQ ID NO: 114.

SEQ ID NO: 116 corresponds to the nucleotide sequence of a first guideRNA target site in the COX1 gene of Saccharomyces cerevisiaemitochondrial DNA. The last three nucleotides are the PAM sequence;these three nucleotides are not present in the variable targeting domainof the corresponding guide RNA.

SEQ ID NO: 117 corresponds to the nucleotide sequence of a second guideRNA target site in the COX1 gene of Saccharomyces cerevisiaemitochondrial DNA. The last three nucleotides are the PAM sequence;these three nucleotides are not present in the variable targeting domainof the corresponding guide RNA.

SEQ ID NO: 118 corresponds to the nucleotide sequence of a third guideRNA target site in the COX1 gene of Saccharomyces cerevisiaemitochondrial DNA. The last three nucleotides are the PAM sequence;these three nucleotides are not present in the variable targeting domainof the corresponding guide RNA.

SEQ ID NO: 119 corresponds to the nucleotide sequence of a fourth guideRNA target site in the COX1 gene of Saccharomyces cerevisiaemitochondrial DNA. The last three nucleotides are the PAM sequence;these three nucleotides are not present in the variable targeting domainof the corresponding guide RNA. This target site sequence is present onthe reverse complement of the genic sequence.

SEQ ID NO: 120 corresponds to the nucleotide sequence encoding SpCas9,the Cas9 from Streptococcus pyogenes. The coding sequence was optimizedfor expression in yeast mitochondria.

SEQ ID NO: 121 corresponds to the nucleotide sequence of the minimalpromoter and 5′ UTR of the COX2 gene of Saccharomyces cerevisiaemitochondrial DNA.

SEQ ID NO: 122 corresponds to the nucleotide sequence of the minimalterminator of the COX2 gene of Saccharomyces cerevisiae mitochondrialDNA.

SEQ ID NO: 123 corresponds to the nucleotide sequence encoding thetracrRNA, which was used to create guide RNAs targeting the COX2 gene ofSaccharomyces cerevisiae.

SEQ ID NO: 124 corresponds to the nucleotide sequence of the minimalpromoter of the COX3 gene of Saccharomyces cerevisiae mitochondrial DNA.

SEQ ID NO: 125 corresponds to the nucleotide sequence encoding the tRNAof the tF(GAA) gene from Saccharomyces cerevisiae mitochondrial DNA.

SEQ ID NO: 126 corresponds to the nucleotide sequence encoding the tRNAof the tW(UCA) gene from Saccharomyces cerevisiae mitochondrial DNA.

SEQ ID NO: 127 corresponds to the nucleotide sequence of the minimalterminator of the COX3 gene from Saccharomyces cerevisiae mitochondrialDNA.

SEQ ID NO: 128 corresponds to the nucleotide sequence encoding the tRNAof the tM(CAU) gene from Saccharomyces cerevisiae mitochondrial DNA.

SEQ ID NO: 129 corresponds to the nucleotide sequence encoding GFP. Thecoding sequence was optimized for expression in yeast mitochondria.

SEQ ID NO: 130 corresponds to the nucleotide sequence encoding thehomologous region from Saccharomyces cerevisiae, designated HR1, whichis adjacent to the first guide RNA target site (SEQ ID NO: 116) in theCOX1 gene.

SEQ ID NO: 131 corresponds to the nucleotide sequence encoding thehomologous region from Saccharomyces cerevisiae, designated HR2, whichis adjacent to the second guide RNA target site (SEQ ID NO: 117) in theCOX1 gene.

SEQ ID NO: 132 corresponds to the nucleotide sequence encoding thehomologous region from Saccharomyces cerevisiae, designated HR3, whichis adjacent to the third guide RNA target site (SEQ ID NO: 118) in theCOX1 gene.

SEQ ID NO: 133 corresponds to the nucleotide sequence encoding thehomologous region from Saccharomyces cerevisiae, designated HR4, whichis adjacent to the fourth guide RNA target site (SEQ ID NO: 119) in theCOX1 gene.

SEQ ID NO: 134 corresponds to the nucleotide sequence present in thedonor DNA that encodes a variant of the first guide RNA target site (SEQID NO: 116) in the COX1 gene. Seven nucleotides have been changed in thevariant.

SEQ ID NO: 135 corresponds to the nucleotide sequence present in thedonor DNA that encodes a variant of the second guide RNA target site(SEQ ID NO: 117) in the COX1 gene. Sixteen nucleotides at the 5′ endhave been deleted in the variant.

SEQ ID NO: 136 corresponds to the nucleotide sequence present in thedonor DNA that encodes a variant of the third guide RNA target site (SEQID NO: 118) in the COX1 gene. Five nucleotides at the 3′ end have beendeleted in the variant.

SEQ ID NO: 137 corresponds to the nucleotide sequence present in thedonor DNA that encodes a variant of the fourth guide RNA target site(SEQ ID NO: 119) in the COX1 gene. Seventeen nucleotides at the 3′ endhave been deleted in the variant.

SEQ ID NO: 138 corresponds to the nucleotide sequence of PCR primer C,present in the COX1 gene of Saccharomyces cerevisiae.

SEQ ID NO: 139 corresponds to the nucleotide sequence of PCR primer D,present in the COX1 gene of Saccharomyces cerevisiae.

SEQ ID NO: 140 corresponds to the nucleotide sequence of PCR primer E,present in the COX1 gene of Saccharomyces cerevisiae.

SEQ ID NO: 141 corresponds to the nucleotide sequence of PCR primer F,present in the COX1 gene of Saccharomyces cerevisiae.

SEQ ID NO: 142 corresponds to the nucleotide sequence of PCR primer 11,present in the GFP coding region of the donor DNA.

SEQ ID NO: 143 corresponds to the nucleotide sequence of PCR primer 12,present in the GFP coding region of the donor DNA.

SEQ ID NO: 144 corresponds to the nucleotide sequence derived from thePCR amplification products of the GFP integration region in transformedyeast mitochondrial DNA.

SEQ ID NO: 145 corresponds to the nucleotide sequence of a first guideRNA target site in the psaA gene of Chlamydomonas reinhardtii plastidDNA. The last three nucleotides are the PAM sequence; these threenucleotides are not present in the variable targeting domain of thecorresponding guide RNA.

SEQ ID NO: 146 corresponds to the nucleotide sequence of a second guideRNA target site in the psaA gene of Chlamydomonas reinhardtii plastidDNA. The last three nucleotides are the PAM sequence; these threenucleotides are not present in the variable targeting domain of thecorresponding guide RNA. This target site sequence is present on thereverse complement of the genic sequence.

SEQ ID NO: 147 corresponds to the nucleotide sequence of a third guideRNA target site in the psaA gene of Chlamydomonas reinhardtii plastidDNA. The last three nucleotides are the PAM sequence; these threenucleotides are not present in the variable targeting domain of thecorresponding guide RNA.

SEQ ID NO: 148 corresponds to the nucleotide sequence of a fourth guideRNA target site in the psaA gene of Chlamydomonas reinhardtii plastidDNA. The last three nucleotides are the PAM sequence; these threenucleotides are not present in the variable targeting domain of thecorresponding guide RNA. This target site sequence is present on thereverse complement of the genic sequence.

SEQ ID NO: 149 corresponds to the nucleotide sequence encoding SpCas9,the Cas9 from Streptococcus pyogenes. The coding sequence wascodon-optimized for expression in Chlamydomonas chloroplasts.

SEQ ID NO: 150 corresponds to the amino acid sequence of SpCas9, theCas9 from Streptococcus pyogenes, which is encoded by the nucleotidesequences of SEQ ID NO: 150 and SEQ ID NO: 120.

SEQ ID NO: 151 corresponds to the nucleotide sequence of the promoterand 5′ UTR of the psaA-exon 1 gene of Chlamydomonas reinhardtii plastidDNA.

SEQ ID NO: 152 corresponds to the nucleotide sequence of the promoterand 5′ UTR of the psbD gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 153 corresponds to the nucleotide sequence of the terminatorof the rbcL gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 154 corresponds to the nucleotide sequence of the promoter ofthe trnW gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 155 corresponds to the nucleotide sequence of the 3′ UTR ofthe trnW gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 156 corresponds to the nucleotide sequence encoding the tRNAof the trnW gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 157 corresponds to the nucleotide sequence encoding the tRNAof the trnK gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 158 corresponds to the nucleotide sequence encoding the tRNAof the trnL gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 159 corresponds to the nucleotide sequence encoding the aadAselectable marker.

SEQ ID NO: 160 corresponds to the nucleotide sequence of the promoterand 5′ UTR of the rbcL gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 161 corresponds to the nucleotide sequence of the 3′ UTR ofthe psbA gene of Chlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 162 corresponds to the nucleotide sequence encoding GFP. Thecoding sequence was codon-optimized for expression in Chlamydomonaschloroplasts.

SEQ ID NO: 163 corresponds to the nucleotide sequence encoding HR1, ahomologous region from Chlamydomonas reinhardtii plastid DNA, that ispresent in a donor DNA.

SEQ ID NO: 164 corresponds to the nucleotide sequence encoding HR2, ahomologous region from Chlamydomonas reinhardtii plastid DNA, that ispresent in a donor DNA.

SEQ ID NO: 165 corresponds to the nucleotide sequence encoding HR3, ahomologous region from Chlamydomonas reinhardtii plastid DNA, that ispresent in a donor DNA.

SEQ ID NO: 166 corresponds to the nucleotide sequence encoding HR4, ahomologous region from Chlamydomonas reinhardtii plastid DNA, that ispresent in a donor DNA.

SEQ ID NO: 167 corresponds to the nucleotide sequence of the forwardprimer of Primer Set 1 (PS1 FWD Primer), designed to amplify 852 bp ofthe GFP integration region in the transformed Chlamydomonas reinhardtiiplastid DNA. PS1 FWD Primer is a chloroplast genomic region-specificprimer.

SEQ ID NO: 168 corresponds to the nucleotide sequence of the reverseprimer of Primer Set 1 (PS1 REV Primer), designed to amplify 852 bp ofthe GFP integration region in the transformed Chlamydomonas reinhardtiiplastid DNA. PS1 REV Primer is a GFP gene-specific primer.

SEQ ID NO: 169 corresponds to the nucleotide sequence of the forwardprimer of Primer Set 2 (PS2 FWD Primer), designed to amplify 712 bp ofthe GFP integration region in the transformed Chlamydomonas reinhardtiiplastid DNA. PS2 FWD Primer is a GFP gene-specific primer.

SEQ ID NO: 170 corresponds to the nucleotide sequence of the reverseprimer of Primer Set 2 (PS2 REV Primer), designed to amplify 712 bp ofthe GFP integration region in the transformed Chlamydomonas reinhardtiiplastid DNA. PS2 REV Primer is a chloroplast genomic region-specificprimer.

SEQ ID NO: 171 corresponds to the nucleotide sequence derived from thePCR amplification products of the GFP integration region in transformedChlamydomonas reinhardtii plastid DNA.

SEQ ID NO: 172 corresponds to the amino acid sequence of a permeantpeptide derived from the third alpha helix of Drosophila melanogastertranscription factor Antennapaedia.

DETAILED DESCRIPTION

The present disclosure now will be described more fully hereinafter butshould not be construed as limited to the embodiments set forth herein.

The meaning of abbreviations can be as follows: “sec” can meansecond(s), “min” can mean minute(s), “h” can mean hour(s), “d” can meanday(s), “μL” can mean microliter(s), “ml” can mean milliliter(s), “L”can mean liter(s), “μM” can mean micromolar, “mM” can mean millimolar,“M” can mean molar, “mmol” can mean millimole(s), “μmole” can meanmicromole(s), “g” can mean gram(s), “μg” can mean microgram(s), “ng” canmean nanogram(s), “U” can mean unit(s), “nt” can mean nucleotide(s);“bp” can mean base pair(s), “kb” can mean kilobase(s) and “kbp” can meankilobase pair(s).

“Transgenic” can refer to any cell, cell line, callus, tissue, organismpart or whole organism (e.g., plant), the genome of which has beenaltered by the presence of a heterologous nucleic acid, such as arecombinant DNA construct. Transgenic events can include those createdby sexual crosses or asexual propagation. In some embodiments, the term“transgenic” may not encompass the alteration of the genome (e.g.,chromosomal or extra-chromosomal) by breeding methods or by naturallyoccurring events such as random cross-fertilization, non-recombinantviral infection, non-recombinant bacterial transformation,non-recombinant transposition, or spontaneous mutation. In someembodiments, the term “transgenic” may encompass the alteration of thegenome (e.g., chromosomal or extra-chromosomal) by breeding methods orby naturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

“Genome”, for example, of a cell or whole organism can encompasschromosomal DNA found within the nucleus (nuclear DNA), and organellarDNA (e.g., mitochondrial DNA, plastid DNA) found within subcellularcomponents of the cell. Methods and compositions of the disclosure canbe used for editing of the nuclear genome, organellar genome (e.g.,mitochondria, chloroplasts), or both.

The terms “full complement” and “full-length complement” can be usedinterchangeably herein, and can refer to a complement of a givennucleotide sequence. In some aspects, the complement and the nucleotidesequence comprise of the same number of nucleotides. In some aspects,the complement and the nucleotide sequence can comprise 100%complementary. The complement and the nucleotide sequence can differ inthe number of nucleotides. Complementarity (e.g., between the complementand the nucleotide sequence) can be at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 97%, at least about 98%, at least about99%, or 100%. Complementarity (e.g., between the complement and thenucleotide sequence) can be at most about 10%, at most about 20%, atmost about 30%, at most about 40%, at most about 50%, at most about 60%,at most about 65%, at most about 70%, at most about 75%, at most about80%, at most about 85%, at most about 90%, at most about 95%, at mostabout 97%, at most about 98%, at most about 99%, or 100%.

“Polynucleotide”, “nucleic acid”, “nucleic acid sequence”, “nucleotidesequence”, or “nucleic acid fragment”, which can be usedinterchangeably, can refer to a polymer of a nucleic acid (e.g., RNA,DNA, or both, and analogs thereof) that can be single-stranded ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. Nucleotides (e.g., in their 5′-monophosphate form) canbe referred to by their single letter designation as follows (for RNA orDNA, respectively): “A” for adenylate or deoxyadenylate, “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purine-basednucleotides (A or G), “Y” for pyrimidine-based nucleotides (C or T), “K”for G or T, “H” for A or C or T, “I” for inosine, and “N” for anynucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein”, which canbe used interchangeably herein, can refer to a polymer of amino acidresidues. The terms can apply to amino acid polymers in which one ormore amino acid residue can be, for example, an artificial chemicalanalogue of a corresponding naturally occurring amino acid and/or tonaturally occurring amino acid polymers. The terms “polypeptide”,“peptide”, “amino acid sequence”, and “protein” can be inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

A “functional fragment” of a polynucleotide or polypeptide can refer toany subset of contiguous nucleotides or contiguous amino acids,respectively, in which the original (e.g., wild type) activity (orsubstantially similar activity) of the polynucleotide or polypeptide canbe retained. The terms “functional fragment”, “functional subfragment”,“fragment that is functionally equivalent”, “subfragment that isfunctionally equivalent”, “functionally equivalent fragment” and“functionally equivalent subfragment” can be used interchangeablyherein.

The terms “functional variant”, “variant that is functionallyequivalent” and “functionally equivalent variant” can be usedinterchangeably herein. In the context of a polynucleotide or apolypeptide, these terms can refer to a variant of the nucleic acidsequence or the amino acid sequence, respectively, in which the originalactivity (or substantially similar activity) of the polynucleotide orpolypeptide can be retained. Fragments and variants can be obtained viamethods such as site-directed mutagenesis and synthetic construction.

The activity of the functional fragment or function variant can be, forexample, about: 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,40%, 30%, 20%, 10%, or less than 10% of that of the original (e.g., wildtype) activity.

“RNA transcript” can refer to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complimentary copy of the DNA sequence, it canbe referred to as the primary transcript. A RNA transcript can bereferred to as the mature RNA, for example, when it is a RNA sequencederived from post-transcriptional processing of the primary transcript.

“Messenger RNA” or “mRNA” can refer to the RNA that is without intronsand that can be translated into protein by the cell.

“Sense” RNA can refer to the RNA transcript that includes the mRNA.Sense RNA can be translated into protein within a cell or in vitro.

“Antisense RNA” can refer to an RNA transcript that can be complementaryto all or part of a target RNA (e.g., a primary transcript or mRNA).Antisense RNA can be used to block expression of a target gene. Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e., at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence. “Functional RNA” can refer toantisense RNA, ribozyme RNA, or other RNA that may not be translated butyet can have an effect on cellular processes. The terms “complement” and“reverse complement” can be used interchangeably herein, for example,with respect to mRNA transcripts and may be used to define the antisenseRNA of the message.

“cDNA” can refer to a DNA that can be complementary to and synthesizedfrom a mRNA template using the enzyme reverse transcriptase. The cDNAcan be single-stranded or converted into the double-stranded form usingthe Klenow fragment of DNA polymerase I.

“Coding region” can refer to the portion of a messenger RNA (or thecorresponding portion of another nucleic acid molecule such as a DNAmolecule) which can encode a protein or polypeptide. “Non-coding region”can refer to a portion of a messenger RNA or other nucleic acid moleculethat are not a coding region, including but not limited to, for example,the promoter region, 5′ untranslated region (“UTR”), 3′ UTR, intron andterminator. The terms “coding region” and “coding sequence” can be usedinterchangeably herein. The terms “non-coding region” and “non-codingsequence” can be used interchangeably herein.

“Coding sequence” can be abbreviated “CDS”. “Open reading frame” can beabbreviated “ORF”.

An “Expressed Sequence Tag” (“EST”) can be a DNA sequence derived from acDNA library. An EST can be a sequence which has been transcribed. AnEST can be obtained by a single sequencing pass of a cDNA insert. Thesequence of an entire cDNA insert can be termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence can be a sequence assembled fromtwo or more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein can be termed a “Complete Gene Sequence”(“CGS”). A CGS can be derived from an FIS or a contig.

“Gene” can refer to a nucleic acid fragment that can express afunctional molecule such as, but not limited to, a specific protein,including: introns, exons, regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” can refer to a gene as found in nature, for example, withits own regulatory sequences.

A “mutated gene” can be a gene that has been altered relative to thecorresponding naturally occurring gene; e.g., through humanintervention. Such a “mutated gene” can have a sequence that differsfrom the sequence of the corresponding non-mutated gene by at least onenucleotide addition, deletion, or substitution. In certain embodimentsof the disclosure, the mutated gene can comprise an alteration thatresults from a polynucleotide guided polypeptide system as disclosedherein. A mutated organism can be an organism comprising a mutated gene;e.g., a mutated plant with an organellar genome comprising a mutatedgene. The terms “mutated gene” and “mutant gene” can be usedinterchangeably herein.

A “silent mutation” can refer to a mutated sequence that has the samefunctionality as the wild-type sequence; e.g., replacement of a codon ina protein-coding region with a synonymous codon that can encode the sameamino acid.

As used herein, a “targeted mutation” can be a DNA modification made ator near a specific target site in the genome. The targeted mutation maybe as small as a single nucleotide change in a native gene. The targetedmutation may involve a larger DNA modification such as the insertion ofone or more heterologous DNAs; e.g., a heterologous regulatory element,a heterologous protein-coding sequence, or an expression cassette codingfor a heterologous protein or functional RNA. The targeted mutation mayalso involve a change in the sequence of a target site.

The term “SDN” can refer to “site-directed nuclease”. The following arenon-limiting examples of SDN-induced mutations: (1) induction ofsite-specific random mutations; (2) the induction of mutations in apredefined sequence of a particular gene; and (3) the replacement or theinsertion of an entire gene. These SDN-induced mutations can be referredto as SDN-1, SDN-2 and SDN-3, respectively.

A “codon-modified gene” or “codon-preferred gene” or “codon-optimizedgene” can be a gene having its frequency of codon usage designed tomimic the frequency of preferred codon usage of the host cell in thecompartment of interest, e.g., the nucleus, the mitochondria or thechloroplast.

“Mature” protein can refer to a post-translationally processedpolypeptide; for example, one from which any pre- or pro-peptidespresent in the primary translation product have been removed.

“Precursor” protein can refer to the primary product of translation ofan mRNA; for example, with pre- and pro-peptides still present. Pre- andpro-peptides may, for example, comprise intracellular localizationsignals.

“Isolated” can refer to materials, such as nucleic acid molecules,proteins, and cells that may be substantially free or otherwise removedfrom components that normally accompany or interact with the materialsin a naturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Nucleic acidpurification methods can be used to obtain isolated polynucleotides.Isolated polynucleotides can include, for example, recombinantpolynucleotides and chemically synthesized polynucleotides.

“Heterologous”, for example, with respect to sequence, can mean asequence that originates from a foreign species, or, if from the samespecies, is substantially modified from its native form in compositionand/or genomic locus by deliberate human intervention. The terms“heterologous nucleotide sequence”, “heterologous sequence”,“heterologous nucleic acid fragment”, and “heterologous nucleic acidsequence” can be used interchangeably herein.

“Recombinant” can refer to an artificial combination of two or moreotherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” can also include reference to acell or vector, for example, that has been modified by the introductionof a heterologous nucleic acid or a cell derived from a cell somodified.

“Recombinant DNA construct” can refer to a combination of nucleic acidfragments that may not normally be found together in nature. Arecombinant DNA construct may comprise, for example, regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource. The sequences in a recombinant DNA construct can be arranged ina manner different than that normally found in nature. The terms“recombinant DNA construct”, “recombinant DNA molecule”, “recombinantconstruct”, “DNA construct” and “construct” can be used interchangeablyherein.

“Expression” can refer to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Expression cassette” can refer to a construct containing, for example,a polynucleotide, a regulatory element(s), and a polynucleotide thatallow for expression of the polynucleotide in a host. The terms“expression cassette” and “expression construct” can be usedinterchangeably herein.

The terms “entry clone” and “entry vector” can be used interchangeablyherein.

“Regulatory sequences” can refer to nucleotide sequences, for example,located upstream (e.g., 5′ non-coding sequences), within (e.g., inintrons), or downstream (e.g., 3′ non-coding sequences) of a codingsequence. Regulatory sequences can influence, for example, thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences may include, but arenot limited to, promoters, translation leader sequences, 5′ untranslatedsequences, 3′ untranslated sequences, introns, polyadenylation targetsequences, RNA processing sites, effector binding sites, and stem-loopstructures. A regulatory sequence may act in “cis” or “trans”. Thenucleic acid molecule regulated by a regulatory sequence may notnecessarily have to encode a functional peptide or polypeptide, e.g.,the regulatory sequence can modulate the expression of a shortinterfering RNA or an anti-sense RNA. The terms “regulatory sequence”and “regulatory element” can be used interchangeably herein.

“Promoter” can refer to a nucleic acid fragment that can controltranscription of another nucleic acid fragment. A promoter can include acore promoter (also known as minimal promoter) sequence. A core promotercan be a minimal sequence for direct transcription initiation. A corepromoter can optionally include enhancers or other regulatory elements.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions.

“Promoter functional in a plant” can be a promoter that can controltranscription in plant cells. The promoter can be from any suitableorigin, which can include plant cells and non-plant cells.

“Tissue-specific promoter” and “tissue-preferred promoter” can be usedinterchangeably, and can refer to a promoter that can be expressedpredominantly in one tissue, one organ or one cell type. Atissue-specific promoter may not be necessarily exclusive in one tissue,one organ or one cell type. Root-preferred promoters include, forexample, the following: soybean root-specific glutamine synthase gene;cytosolic glutamine synthase (GS); root-specific control element in theGRP 1.8 gene of French bean; root-specific promoter of A. tumefaciensmannopine synthase (MAS); root-specific promoters isolated fromParasponia andersonii and Trema tomentosa; A. rhizogenes rolC and rolDroot-inducing genes; Agrobacterium wound-induced TR1′ and TR2′ genes;VfENOD-GRP3 gene promoter; and rolB promoter. Seed-preferred promotersinclude both seed-specific promoters active during seed development, aswell as seed-germinating promoters active during seed germination.Seed-preferred promoters include, but are not limited to, the following:Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase); END1; and END2. For dicots,seed-preferred promoters include, but are not limited to, the following:bean β-phaseolin; napin; β-conglycinin; soybean lectin; cruciferin; andthe like. For monocots, seed-preferred promoters include, but are notlimited to, the following: maize 15 kDa zein; 22 kDa zein; 27 kDa gammazein; waxy; shrunken 1; shrunken 2; globulin 1; oleosin; nud; and Zeamays-Rootmet2 promoter. Leaf-preferred promoters include, but are notlimited to, the following: plant rbcS promoters, such as the soybeanrbcS promoter and the maize rbcS promoter; Zea mays PEPC1 promoter.

“Developmentally regulated promoter” can refer to a promoter whoseactivity can be determined by developmental events.

“Inducible promoter” can refer to a promoter that selectively expressesan operably linked DNA sequence in response to the presence of anendogenous or exogenous stimulus, for example by chemical compounds(e.g., chemical inducers) or in response to environmental, hormonal,chemical, and/or developmental signals. Inducible or regulated promotersinclude, for example, promoters regulated by light, heat, stress,flooding or drought, phytohormones, wounding, or chemicals such asethanol, jasmonate, salicylic acid, or safeners. Pathogen-induciblepromoters induced following infection by a pathogen include, but are notlimited to those regulating expression of PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. Stress-inducible promoters includeplant RAB17 promoters, such as the maize RAB17 promoter.Chemical-inducible promoters include, but are not limited to, thefollowing: the maize ln2-2 promoter, activated by benzene sulfonamideherbicide safeners; the maize GST promoter, activated by hydrophobicelectrophilic compounds used as pre-emergent herbicides; and the tobaccoPR-la promoter, activated by salicylic acid. Other chemical-regulatedpromoters include steroid-responsive promoters, for example, theglucocorticoid-inducible promoter, and tetracycline-inducible andtetracycline-repressible promoters.

“Constitutive promoter” can refer to promoters active in all or mosttissues or cell types of an organism at all or most developing stages.As with other promoters classified as “constitutive” (e.g. ubiquitin),some variation in absolute levels of expression can exist amongdifferent tissues or stages. The term “constitutive promoter” or“tissue-independent promoter” can be used interchangeably herein.Constitutive promoters include the following: the core promoter of theRsyn7 promoter; the core CaMV 35S promoter; plant actin promoter, suchas a rice actin promoter and a maize actin promoter; plant ubiquitinpromoter, such as a maize ubiquitin promoter and a soybean ubiquitinpromoter; pEMU; MAS promoter; ALS promoter; plant GOS2 promoter, such asa maize GOS2 promoter; soybean GM-EF1 A2 promoter; plant U6 polymeraseIII promoter, such as a maize U6 polymerase III promoter and a soybeanU6 polymerase III promoter (GM-U6-9.1 and GM-U6-13.1).

An enhancer element can be any nucleic acid molecule that increasestranscription of a nucleic acid molecule when functionally linked to apromoter regardless of its relative position. An enhancer may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter.

A repressor (also sometimes called herein silencer) can be defined asany nucleic acid molecule which inhibits the transcription whenfunctionally linked to a promoter regardless of relative position.

“Translation leader sequence” can refer to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence can be present in the fully processedmRNA upstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency.

“Transcription terminator”, “termination sequence”, or “terminator” canrefer to DNA sequences that, when operably linked to the 3′ end of apolynucleotide sequence that is to be expressed, can terminatetranscription from the polynucleotide sequence. Transcriptiontermination can refer to the process by which RNA synthesis by RNApolymerase can be stopped and both the RNA and the enzyme are releasedfrom the DNA template.

“Operably linked” can refer to the association of fragments in a singlefragment (e.g., a polynucleotide or polypeptide), or in a singlecomplex, so that the function of one can be regulated by the other. Thelinkage may be covalent or non-covalent. For example, with respect tonucleic acid fragments, a promoter can be operably linked with a nucleicacid fragment if the promoter can regulate the transcription of thatnucleic acid fragment. For example, with respect to a polypeptide, anorganelle targeting peptide can be operably linked with a polypeptide ifthe organelle targeting peptide can transport that polypeptide into therelevant organelle. For example, with respect to a complex, a guide RNAcan be operably linked to a Cas polypeptide if the guide RNA/Caspolypeptide complex can cleave a target sequence as directed by theguide RNA.

“Phenotype” can refer to the detectable characteristics of a cell ororganism.

The term “introduced” can mean providing a polynucleic acid (e.g.,expression construct) or protein into a cell. Introduced can includereference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell, for example, where the nucleic acid may beincorporated into the genome of the cell. Introduced can includereference to the transient provision of a nucleic acid or protein to thecell. Introduced can include reference to stable or transienttransformation methods. Introduced can include sexually crossing.Introduced, for example, in the context of inserting a nucleic acidfragment (e.g., a recombinant DNA construct) into a cell, can include“transfection” or “transformation” or “transduction”. Introduced caninclude reference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” can be any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein can refer to stable transformation.Transformation can refer to transient transformation.

“Stable transformation” can refer to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentcan be stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” can refer to the introduction of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

Host organisms containing the transformed nucleic acid fragments can bereferred to as “transgenic” organisms.

“Transformation cassette” can refer to a construct having elements thatfacilitates transformation of a particular host cell. The terms“transformation cassette” and “transformation construct” can be usedinterchangeably herein.

“Allele” can be one of several alternative forms of a gene occupying agiven locus on a chromosome. When the alleles present at a given locuson a pair of homologous chromosomes in a diploid plant are the same thatplant can be homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ,that plant can be heterozygous at that locus. If a transgene is presenton one of a pair of homologous chromosomes in a diploid plant that plantcan be hemizygous at that locus.

A “chloroplast transit peptide” can be an amino acid sequence that candirect a protein to the chloroplast or other plastid types present inthe cell. The chloroplast transit peptide can be translated inconjunction with the protein in the cell in which the protein can bemade. The terms “chloroplast transit peptide”, “plastid transitpeptide”, “chloroplast targeting peptide” and “plastid targetingpeptide” can be used interchangeably herein. “Chloroplast transitsequence” can refer to a nucleotide sequence that can encode achloroplast transit peptide.

A “signal peptide” can be an amino acid sequence that can direct aprotein to the secretory system. The signal peptide can be translated inconjunction with a protein. For example, if the protein is to bedirected to a vacuole, a vacuolar targeting signal (supra) can furtherbe added, or if to the endoplasmic reticulum, an endoplasmic reticulumretention signal (supra) may be added. If the protein is to be directedto the nucleus, any signal peptide present may be removed and a nuclearlocalization signal can be included.

A “mitochondrial signal peptide” can be an amino acid sequence which candirect a precursor protein into the mitochondria. The terms“mitochondrial signal peptide”, “mitochondrial transit peptide” and“mitochondrial targeting peptide” can be used interchangeably herein.

An “organelle targeting polynucleotide” can be a nucleotide sequencewhich can direct import of the polynucleotide into an organelle. Theterms “organelle targeting polynucleotide”, “organelle targeting nucleicacid” and “organelle targeting nucleic acid sequence” can be usedinterchangeably herein. An organelle targeting polynucleotide may bedirected to, for example, the plastid (“plastid targetingpolynucleotide”) or the mitochondria (“mitochondria targetingpolynucleotide”). The polynucleotide may be RNA (“organelle targetingRNA”), DNA (“organelle targeting DNA) or a combination of RNA and DNA.An organelle targeting RNA directed to the plastid can be termed a“plastid targeting RNA”. The terms “plastid targeting RNA”, “chloroplasttargeting RNA” and “transit RNA” are used interchangeably herein. Anorganelle targeting RNA directed to the mitochondria can be termed a“mitochondria targeting RNA”.

RNAs can be imported into mitochondria. One such mitochondrial targetingRNA can be the yeast tRNA^(Lys). The yeast tRNA^(Lys) and its variantscan be imported into human mitochondria. Another RNA that can beimported into mitochondria can be 5S rRNA. 5S rRNA can function as avector for delivering heterologous RNA sequences into, for example,mitochondria (e.g., human). Such RNAs can be used with the compositionsand methods of the disclosure for example, for targeting to an organelle(e.g., the mitochondria).

RNAs can be imported into plastids. Plastid targeting RNAs that canmediate import of attached heterologous RNA can include vd-5′UTR (e.g.,viroid-derived ncRNA sequence acting as 5′UTR and eIF4E1 mRNA. Such RNAscan be used with the compositions and methods of the disclosure fortargeting to an organelle (e.g., the plastid).

As used herein, “fusion” can refer to a protein and/or nucleic acidcomprising one or more non-native sequences (e.g., moieties). Any of themolecules described herein (e.g., nucleic acids, proteins, polypeptides,polynucleic acid, Cas protein, guide polynucleotide) can be engineeredas fusions. A fusion can comprise one or more of the same non-nativesequences. A fusion can comprise one or more of different non-nativesequences. A fusion can be a chimera. A fusion can comprise a nucleicacid affinity tag. A fusion can comprise a barcode. A fusion cancomprise a peptide affinity tag. A fusion can provide for subcellularlocalization of the site-directed polypeptide. A fusion can provide anon-native sequence (e.g., affinity tag) that can be used to track orpurify. A fusion can be a small molecule such as biotin or a dye such asalexa fluor dyes, Cyanine3 dye, and Cyanine5 dye.

A fusion can refer to any protein with a functional effect. For example,a fusion protein can comprise deaminase activity, cytidine deaminaseactivity (US Patent Publication No. US20150166980, herein incorporatedby reference), adenine deaminase activity (US Patent Publication No.US20180073012, herein incorporated by reference), uracil glycosylaseinhibitor activity (US Patent Publication No. US20170121693, hereinincorporated by reference), methyltransferase activity, demethylaseactivity, dismutase activity, alkylation activity, depurinationactivity, oxidation activity, pyrimidine dimer forming activity,integrase activity, transposase activity, recombinase activity,polymerase activity, ligase activity, helicase activity, photolyaseactivity or glycosylase activity, acetyltransferase activity,deacetylase activity, kinase activity, phosphatase activity, ubiquitinligase activity, deubiquitinating activity, adenylation activity,deadenylation activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, deribosylation activity, myristoylation activity,remodeling activity, protease activity, oxidoreductase activity,transferase activity, hydrolase activity, lyase activity, isomeraseactivity, synthase activity, synthetase activity, or demyristoylationactivity. An effector protein can modify a genomic locus. A fusionprotein can be a fusion in a Cas protein. The Cas protein may be amodified form that has nickase activity or that has no substantialnucleic acid-cleaving activity. A fusion protein can be a non-nativesequence in a Cas protein.

As used herein, a “nucleic acid” can refer to a polynucleotide sequence,or fragment thereof. A nucleic acid can comprise nucleotides. A nucleicacid can be exogenous or endogenous to a cell. A nucleic acid can existin a cell-free environment. A nucleic acid can be a gene or fragmentthereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleicacid can comprise one or more analogs (e.g. altered backgone, sugar, ornucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamineor flurescein linked to the sugar), thiol containing nucleotides, biotinlinked nucleotides, fluorescent base analogs, CpG islands,methyl-7-guanosine, methylated nucleotides, inosine, thiouridine,pseudourdine, dihydrouridine, queuosine, and wyosine.

Suppression of Gene Expression

“Suppression DNA construct” can be a recombinant DNA construct whichwhen transformed or stably integrated into the genome of the plant, canresult in “silencing” of a target gene (e.g., in a plant). The targetgene may be endogenous or transgenic to a target cell (e.g., plant).

“Silencing,” as used herein with respect to the target gene, can referto the suppression of levels of mRNA or protein/enzyme expressed by thetarget gene, and/or the level of the enzyme activity or proteinfunctionality. The terms “suppression”, “suppressing” and “silencing”,which can be used interchangeably herein, can include lowering,reducing, declining, decreasing, inhibiting, eliminating or preventing.“Silencing” or “gene silencing” can occur by any suitable mechanism.Non-limiting examples of silencing can include anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches

A suppression DNA construct may comprise a region derived from a targetgene of interest. A suppression DNA construct may comprise all or partof the nucleic acid sequence of the sense strand (or antisense strand,or both) of the target gene of interest. The region may be 100%identical or less than 100% identical (e.g., at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand(or antisense strand, or both) of the gene of interest. A suppressionDNA construct may comprise 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of thesense strand (or antisense strand, or both) of the gene of interest, andcombinations thereof.

Suppression DNA constructs can be readily constructed, for example, oncethe target gene of interest is selected. A suppression DNA construct caninclude, without limitation, cosuppression constructs, antisenseconstructs, viral-suppression constructs, hairpin suppressionconstructs, stem-loop suppression constructs, double-strandedRNA-producing constructs, and more generally, RNAi (RNA interference)constructs and small RNA constructs such as siRNA (short interferingRNA) constructs and miRNA (microRNA) constructs.

Suppression of gene expression may also be achieved by, for example, useof artificial miRNA precursors, ribozyme constructs and gene disruption.A modified plant miRNA precursor may be used, wherein the precursor hasbeen modified, for example, to replace the miRNA encoding region with asequence designed to produce a miRNA directed to the nucleotide sequenceof interest. Gene disruption may be achieved by use of transposableelements or by use of chemical agents that cause site-specificmutations.

“Antisense inhibition” can refer to the production of antisense RNAtranscripts that can suppress the expression of the target gene or geneproduct. “Antisense RNA” can refer to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA.Antisense RNA can block the expression of a target isolated nucleic acidfragment. The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence.

“Cosuppression” can refer to the production of sense RNA transcriptsthat can suppress the expression of the target gene or gene product.“Sense” RNA can refer to RNA transcript that can include the mRNA. SenseRNA can be translated into protein within a cell or in vitro.Cosuppression constructs in plants can be designed, for example, byfocusing on overexpression of a nucleic acid sequence having homology toa native mRNA, in the sense orientation, which can result in thereduction of RNA having homology to the overexpressed sequence.

Plant viral sequences can be used to direct the suppression of proximalmRNA encoding sequences.

RNA interference can refer to the process of sequence-specificpost-transcriptional gene silencing (e.g., in animals) mediated by, forexample, short interfering RNAs (siRNAs). The corresponding process inplants can be referred to as post-transcriptional gene silencing (PTGS)or RNA silencing and can also referred to as quelling in fungi. Theprocess of post-transcriptional gene silencing can be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes. Post-transcriptional gene silencing can beshared by diverse flora and phyla.

Small RNAs can play an important role in controlling gene expression.Small RNAs can function by base-pairing to complementary RNA or DNAtarget sequences. When bound to RNA, small RNAs can trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, small RNAs can mediate DNA methylation of thetarget sequence. Small RNAs can lead to inhibition of gene expression.

MicroRNAs (miRNAs) can be noncoding RNAs with a length of, for example,about 19 to about 24 nucleotides (nt). MicroRNAs can occur in animalsand plants. miRNAs can be processed from longer precursor transcriptsthat can range in size, for example, from approximately 70 to 200 nt.The precursor transcripts can form stable hairpin structures.

MicroRNAs (miRNAs) can regulate target genes, for example, by binding tocomplementary sequences located in the transcripts produced by thesegenes. miRNAs can enter, for example, at least two pathways of targetgene regulation: (1) translational inhibition; and/or (2) RNA cleavage.MicroRNAs entering the RNA cleavage pathway can be analogous to the21-25 nt short interfering RNAs (siRNAs) generated during RNAinterference (RNAi) in animals and posttranscriptional gene silencing(PTGS) in plants. These microRNAs entering the RNA cleavage pathway canbe incorporated into an RNA-induced silencing complex (RISC) that can besimilar or identical to that seen for RNAi.

The terms “miRNA-star sequence” and “miRNA* sequence” can be usedinterchangeably herein and can refer to a sequence in the miRNAprecursor that can be highly complementary to the miRNA sequence. ThemiRNA and miRNA* sequences can form part of the stem region of the miRNAprecursor hairpin structure.

Sequence Identity, Similarity and Variation

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MEGALIGN™program of the LASERGENE™ bioinformatics computing suite (DNASTAR™ Inc.,Madison, Wis.). In some embodiments, where sequence analysis software isused for analysis, the results of the analysis can be based on the“default values” of the program referenced. As used herein “defaultvalues” can mean any set of values or parameters that originally loadwith the software when first initialized.

The “Clustal V method of alignment” can correspond to the alignmentmethod labeled Clustal V and, for example, found in the MEGALIGN™program of the LASERGENE™ bioinformatics computing suite (DNASTAR™ Inc.,Madison, Wis.). For multiple alignments, the default values cancorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method can be, for example,KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters can be for example KTUPLE=2, GAP PENALTY=5,WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences usingthe Clustal V program, “percent identity” and “divergence” values can beobtained by viewing the “sequence distances” table in the same program.

The “Clustal W method of alignment” can correspond to the alignmentmethod labeled Clustal W and, for example, found in the MEGALIGN™ v6.1program of the LASERGENE™ bioinformatics computing suite (DNASTAR™ Inc.,Madison, Wis.). Default parameters for multiple alignment can correspondto for example: GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay DivergenceSequences=30%, DNA Transition Weight=0.5, Protein Weight Matrix=GonnetSeries, DNA Weight Matrix=IUB. After alignment of the sequences usingthe Clustal W program, “percent identity” values can be obtained byviewing the “sequence distances” table in the same program.

Sequence identity/similarity values can also be obtained using GAPVersion 10 (GCG, Accelrys, San Diego, Calif.) using for example thefollowing parameters: % identity and % similarity for a nucleotidesequence using a gap creation penalty weight of 50 and a gap lengthextension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using a GAPcreation penalty weight of 8 and a gap length extension penalty of 2,and the BLOSUM62 scoring matrix. GAP can use an algorithm to find analignment of two complete sequences that can maximize the number ofmatches and minimizes the number of gaps. GAP can consider all possiblealignments and gap positions. GAP can create the alignment with thelargest number of matched bases and the fewest gaps, using, for example,a gap creation penalty and a gap extension penalty in units of matchedbases.

“BLAST” can be a searching algorithm provided by the National Center forBiotechnology Information (NCBI) that can be used to find regions ofsimilarity between biological sequences. The program can comparenucleotide or protein sequences to sequence databases. The program cancalculate the statistical significance of matches to identify sequenceshaving sufficient similarity to a query sequence such that thesimilarity may not be predicted to have occurred randomly. BLAST canreport the identified sequences and their local alignment to the querysequence.

The term “conserved domain” or “motif” can mean a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions can indicate, for example, amino acidsthat are essential to the structure, the stability, or the activity of aprotein.

Conserved domains or motifs can be identified by their high degree ofconservation in aligned sequences of a family of protein homologues.Conserved domains can be used as identifiers, or “signatures”, forexample, to determine if a protein with a newly determined sequencebelongs to a previously identified protein family.

Polynucleotide and polypeptide sequences, variants thereof, and thestructural relationships of these sequences can be described by theterms “homology”, “homologous”, “substantially identical”,“substantially similar” and “corresponding substantially” which are usedinterchangeably herein. These can refer to polypeptide or nucleic acidfragments wherein changes in one or more amino acids or nucleotide basesmay not affect the function of the molecule, such as the ability tomediate gene expression or to produce a certain phenotype. These termscan also refer to modification(s) of nucleic acid fragments that may notsubstantially alter the functional properties of the resulting nucleicacid fragment relative to the initial, unmodified fragment. Thesemodifications can include deletion, substitution, and/or insertion ofone or more nucleotides in the nucleic acid fragment.

Substantially similar nucleic acid sequences encompassed may be definedby their ability to hybridize (for example, under moderately stringentconditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequencesexemplified herein, or to any portion of the nucleotide sequencesdisclosed herein. Substantially similar nucleic acid sequences can befunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes can determine stringency conditions.

The term “selectively hybridizes” can include reference tohybridization, for example under stringent hybridization conditions, ofa nucleic acid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences can have, for example, about at least 80% sequenceidentity, or 90% sequence identity, up to and including 100% sequenceidentity (i.e., fully complementary) with each other.

The term “stringent conditions” or “stringent hybridization conditions”can include reference to conditions under which a probe can selectivelyhybridize to its target sequence in an in vitro hybridization assay.Stringent conditions can be sequence-dependent. Stringent conditions canbe different in different circumstances. By controlling the stringencyof the hybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing).

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). In some embodiments, a probe can beless than about 1000 nucleotides in length, optionally less than 500nucleotides in length.

In some embodiments, stringent conditions can be those in which the saltconcentration is less than about 1.5 M Na ion, for example, about 0.01to 1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and,for example, at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and, for example, at least about 60° C. for long probes(e.g., greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.Exemplary low stringency conditions can include hybridization with abuffer solution of, for example, 30 to 35% formamide, 1 M NaCl, 1% SDS(sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC(20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplarymoderate stringency conditions can include hybridization in 40 to 45%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55to 60° C. Exemplary high stringency conditions can include hybridizationin, for example, 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1×SSC at 60 to 65° C.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences can refer to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

The term “percentage of sequence identity” can refer to the valuedetermined by comparing two optimally aligned sequences over acomparison window. The portion of the polynucleotide or polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which may or may notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage can be calculated by, for example, determiningthe number of positions at which the identical nucleic acid base oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison and multiplying theresults by 100 to yield the percentage of sequence identity. Percentsequence identities can include, but are not limited to, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%.Sequence identity can include an integer percentage from 50% to 100%.These identities can be determined using any of the programs describedherein.

Sequence identity can be useful in identifying polypeptides from otherspecies or modified naturally or synthetically wherein such polypeptideshave the same or similar function or activity. Percent identities caninclude, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95%. Sequence identity (e.g., amino acid sequence identity) caninclude an integer percentage from 50% to 100%. Sequence (e.g., aminoacid) identity can include, for example, about: 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%.

Definitions, Traits and Processes Relevant to Plants

“Plant” can include reference to whole plants, plant organs, planttissues, plant propagules, seeds and plant cells and progeny of same.Plant cells include, without limitation, cells from seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen, and microspores.

“Propagule” can include products of meiosis and/or mitosis able topropagate a new plant. Propagule can include seeds, spores and parts ofa plant that can serve as a means of vegetative reproduction, such ascorms, tubers, offsets, or runners. Propagule can include grafts whereone portion of a plant can be grafted to another portion of a differentplant (even one of a different species) to create a living organism.Propagule can include plants and seeds produced by cloning or bybringing together meiotic products, or allowing meiotic products to cometogether to form an embryo or fertilized egg (naturally or with humanintervention).

“Progeny” can comprise any subsequent generation of a plant.

The terms “monocot” and “monocotyledonous plant” can be usedinterchangeably herein. A monocot can include the Gramineae.

The terms “dicot” and “dicotyledonous plant” can be used interchangeablyherein. A dicot can include, for example, the following families:Brassicaceae, Leguminosae, and Solanaceae.

“Transgenic plant” can include reference to a plant which compriseswithin its genome a heterologous polynucleotide. For example, theheterologous polynucleotide may be stably integrated within the genome(e.g., nuclear, plastid, mitochondrial) such that the polynucleotide canbe passed on to successive generations. The heterologous polynucleotidemay be integrated into the genome alone or as part of a recombinant DNAconstruct.

“Transgenic plant” can include reference to plants which can comprisemore than one heterologous polynucleotide within their genome. Eachheterologous polynucleotide may confer a different trait to thetransgenic plant.

Multiple traits can be introduced into crop plants, and can be referredto as a gene stacking approach. Gene stacking can be used, for example,for development of genetically improved germplasm. In this approach,multiple genes conferring different characteristics of interest can beintroduced into a plant. Gene stacking can be accomplished by many meansincluding but not limited to co-transformation, retransformation, andcrossing lines with different transgenes. As used herein, the term“stacked” can include having multiple traits present in the same plant(e.g., both traits are incorporated into the nuclear genome, one traitis incorporated into the nuclear genome and one trait is incorporatedinto the genome of an organelle, or both traits are incorporated intothe genome of an organelle).

The term “crossed” or “cross” or “crossing” in the context of thedisclosure can mean the fusion of gametes (e.g., via pollination) toproduce progeny (e.g., cells, seeds, or plants). The term can encompassboth sexual crosses (e.g., the pollination of one plant by another) andselfing (e.g., self-pollination; when the pollen and ovule are from thesame plant or genetically identical plants).

The term “maternal inheritance” can refer to the transmission of traitsthat can be solely dependent on properties of the genome of the femalegamete.

The term “paternal inheritance” can refer to the transmission of traitsthat are solely dependent on properties of the genome of the malegamete.

The term “introgression” can refer to the transmission of a desiredallele of a genetic locus from one genetic background to another. Forexample, introgression of a desired allele at a specified locus can betransmitted to at least one progeny plant via a sexual cross between twoparent plants, where at least one of the parent plants has the desiredallele within its genome. Alternatively, for example, transmission of anallele can occur by recombination between two donor genomes, e.g., in afused protoplast, where at least one of the donor protoplasts has thedesired allele in its genome. The desired allele can be, e.g., atransgene or a selected allele of a marker or QTL.

“A plant-optimized nucleotide sequence” can be a nucleotide sequencethat has been optimized for increased expression in plants, particularlyfor increased expression in plants or in one or more plants of interest.For example, a plant-optimized nucleotide sequence can be synthesized bymodifying a nucleotide sequence encoding a protein such as, for example,a double-strand-break-inducing agent (e.g., an endonuclease) asdisclosed herein, using one or more plant-preferred codons for improvedexpression. A host-preferred codon usage can be utilized for codonoptimization.

Plant-preferred genes can be synthesized. Additional sequencemodifications can enhance gene expression in a plant host. These caninclude, for example, elimination of: one or more sequences encodingspurious polyadenylation signals, one or more exon-intron splice sitesignals, one or more transposon-like repeats, and sequences that may bedeleterious to gene expression. The G-C content of the sequence may beadjusted, for example, to levels average for a given plant host, ascalculated by reference to genes expressed in the host plant cell. Whenpossible, the sequence can be modified to avoid one or more predictedhairpin secondary mRNA structures. Thus, “a plant-optimized nucleotidesequence” of the present disclosure can comprise one or more of suchsequence modifications.

A “trait” can refer to, for example, a physiological, morphological,biochemical, or physical characteristic of a plant or particular plantmaterial or cell. In some instances, this characteristic can be visibleto the human eye, such as seed or plant size, or can be measured bybiochemical techniques, such as detecting the protein, starch, or oilcontent of seed or leaves, or by observation of a metabolic orphysiological process, e.g. by measuring tolerance to water deprivationor particular salt or sugar concentrations, or by the observation of theexpression level of a gene or genes, or by agricultural observationssuch as osmotic stress tolerance or yield.

“Agronomic characteristic” can be a measurable parameter including butnot limited to, abiotic stress tolerance, greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height, ear length, salt tolerance, early seedling vigor andseedling emergence under low temperature stress.

Particular phenotypes may include, but are not limited to kernel number,kernel area, grain weight, and predicted weight of the grain on the ear(based on the calibration of kernel area to grain weight).

Abiotic stress may be at least one condition selected from the groupconsisting of: drought, water deprivation, flood, high light intensity,high temperature, low temperature, salinity, etiolation, defoliation,heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrientexcess, UV irradiation, atmospheric pollution (e.g., ozone) and exposureto chemicals (e.g., paraquat) that induce production of reactive oxygenspecies (ROS).

“Increased stress tolerance” of a plant can be measured relative to areference or control plant, and can be a trait of the plant to surviveunder stress conditions over prolonged periods of time, withoutexhibiting the same degree of physiological or physical deteriorationrelative to the reference or control plant grown under similar stressconditions.

A plant with “increased stress tolerance” can exhibit increasedtolerance to one or more different stress conditions.

“Stress tolerance activity” of a polypeptide can indicate thatover-expression of the polypeptide in a transgenic plant can conferincreased stress tolerance to the transgenic plant relative to areference or control plant.

Increased biomass can be measured, for example, as an increase in plantheight, plant total leaf area, plant fresh weight, plant dry weight orplant seed yield, as compared with control plants.

The ability to increase the biomass or size of a plant can have severalimportant commercial applications. Crop species may be generated thatcan produce larger cultivars, generating higher yield in, for example,plants in which the vegetative portion of the plant can be useful asfood, biofuel or both.

Increased leaf size can be produced by the methods and composition ofthe disclosure. Increasing leaf biomass can be used to increaseproduction of plant-derived pharmaceutical or industrial products. Anincrease in total plant photosynthesis can be achieved by, for example,increasing leaf area of the plant. Additional photosynthetic capacitymay be used to increase the yield derived from particular plant tissue,including the leaves, roots, fruits or seed, or permit the growth of aplant under decreased light intensity or under high light intensity.

Modification of the biomass of a tissue, such as root tissue, may beuseful to improve a plant's ability to grow under harsh environmentalconditions, including drought or nutrient deprivation. Larger roots maybetter reach water or nutrients or take up water or nutrients.

The ability to provide larger varieties can be highly desirable, forexample, for some ornamental plants. For many plants, includingfruit-bearing trees, trees that are used for lumber production, or treesand shrubs that serve as view or wind screens, increased stature canprovide improved benefits in the forms of greater yield or improvedscreening.

Herbicide Resistance in Plants

An “herbicide resistance protein” or a protein resulting from expressionof an “herbicide resistance-encoding nucleic acid molecule” can includeproteins that can confer upon a cell the ability to tolerate a higherconcentration of an herbicide, for example, compared with cells that donot express the protein. An herbicide resistance protein or a proteinresulting from expression of a herbicide resistance-encoding nucleicacid molecule can include proteins that can confer upon a cell theability to tolerate a concentration of a herbicide for a longer periodof time than cells that do not express the protein. Herbicide resistancetraits may be introduced into plants by, for example, genes coding forresistance to herbicides. Genes coding for resistance to herbicidesinclude, for example, genes that act to inhibit the action ofacetolactate synthase (ALS), such as the sulfonylurea-type herbicides,genes that act to inhibit the action of glutamine synthase, such asphosphinothricin or basta (e.g., the bar gene), glyphosate (e.g., theEPSP synthase gene), HPPD inhibitors (e.g., the HPPD gene).

Herbicide resistance proteins can include the following: a4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide and an acetyl coenzyme Acarboxylase (ACCase). Non-limiting examples of genes useful forconferring herbicide resistance in plants can include genes that encodethe above proteins.

As used herein, “Hydroxyphenylpyruvate dioxygenase” and “HPPD”,“4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4-HPPD)” and“p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (p-OHPP)” canbe synonymous and can refer to a non-heme iron-dependent oxygenase thatcatalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. Inorganisms that degrade tyrosine, the reaction catalyzed by HPPD can bethe second step in the pathway. In plants, formation of homogentisatecan be necessary for the synthesis of plastoquinone, which can serve asa redox cofactor, and tocopherol. A polynucleotide molecule encodinghydroxyphenylpyruvate dioxygenase (HPPD) can provide tolerance to HPPDinhibitors.

As used herein, an “HPPD inhibitor” can comprise any compound orcombinations of compounds which can decrease the ability of HPPD tocatalyze the conversion of 4-hydroxyphenylpyruvate to homogentisate. Inspecific embodiments, the HPPD inhibitor can comprise an herbicidalinhibitor of HPPD. Non-limiting examples of HPPD inhibitors include,triketones (such as, mesotrione, sulcotrione, topramezone, andtembotrione); isoxazoles (such as, pyrasulfotole and isoxaflutole);pyrazoles (such as, benzofenap, pyrazoxyfen, and pyrazolynate); andbenzobicyclon. Agriculturally acceptable salts of the various inhibitorscan include salts (e.g., the cations or anions) for the formation ofsalts for agricultural or horticultural use.

An “ALS inhibitor-tolerant polypeptide” can comprise any polypeptidewhich when expressed in a plant can confer tolerance to at least one ALSinhibitor. ALS inhibitors include, for example, sulfonylurea,imidazolinone, triazolopyrimidines, pryimidinyoxy(thio)benzoates, and/orsulfonylaminocarbonyltriazolinone herbicides. ALS mutations can fallinto different classes with regard to tolerance to, for example,sulfonylureas, imidazolinones, triazolopyrimidines, andpyrimidinyl(thio)benzoates. ALS mutations can include mutations havingone or more of the following characteristics: (1) broad tolerance to allfour of these groups (e.g., sulfonylureas, imidazolinones,triazolopyrimidines, and pyrimidinyl(thio)benzoates); (2) tolerance toimidazolinones and pyrimidinyl(thio)benzoates; (3) tolerance tosulfonylureas and triazolopyrimidines; and (4) tolerance tosulfonylureas and imidazolinones.

Polynucleotide molecules encoding proteins involved in herbicideresistance can include a polynucleotide molecule encoding5-enolpymvylshikimate-3-phosphate synthase (EPSPS) for example, forimparting glyphosate tolerance.

Glyphosate tolerance can also be obtained by expression ofpolynucleotide molecules encoding a glyphosate oxidoreductase (GOX) or aglyphosate-N-acetyl transferase (GAT).

Polynucleotides encoding an exogenous phosphinothricin acetyltransferasecan be used for herbicide resistance. Plants containing an exogenousphosphinothricin acetyltransferase can exhibit improved tolerance toglufosinate herbicides, which can inhibit, for example, the enzymeglutamine synthase.

Polynucleotides conferring altered protoporphyrinogen oxidase (protox)activity can be used for herbicide resistance. Plants containing suchpolynucleotides can exhibit improved tolerance to any of a variety ofherbicides which can target, for example, the protox enzyme (alsoreferred to as “protox inhibitors”).

Dicamba monooxygenase can be used for providing dicamba tolerance.

A polynucleotide molecule encoding AAD12 or encoding AAD1 can be usedfor providing resistance to, for example, auxin herbicides.

A P450 sequence can be used for conferring herbicide resistance. A P450sequence can provide tolerance to HPPD inhibitors by, for example,metabolism of the herbicide. Such sequences include, but are not limitedto, the NSF1 gene.

Pest Resistance in Plants by Gene Silencing

A “plant pest” can mean any living stage of an entity that can directlyor indirectly injure, cause damage to, or cause disease in any plant orplant product. A plant pest can include a protozoan, a nonhuman animal,a parasitic plant, a bacterium, a fungus, a virus, a viroid, aninfectious agent, a pathogen, or any article similar to or alliedthereof.

Double-stranded RNA (dsRNA) can be used to provide resistance to plantpests.

Plant pest invertebrates can include, but are not limited to, pestnematodes, pest mollusks (slugs and snails), and pest insects. Plantpathogens can include fungi and nematodes.

The plant pathogen can be a eukaryotic plant pathogen. This includes forexample, a fungal pathogen, such as a phytopathogenic fungus.

Non-limiting examples of fungal plant pathogens include, e.g., the fungithat cause powdery mildew, rust, leaf spot and blight, damping-off, rootrot, crown rot, cotton boll rot, stem canker, twig canker, vascularwilt, smut, or mold, including, but not limited to, Fusarium spp.,Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp.,Pyricularia spp., Alternaria spp., and Phytophthora spp. Specificexamples of fungal plant pathogens include Phakospora pachirhizi (Asiansoy rust), Puccinia sorghi (corn common rust), Puccinia polysora (cornSouthern rust), Fusarium oxysporum and other Fusarium spp., Alternariaspp., Penicillium spp., Pythium aphanidermatum and other Pythium spp.,Rhizoctonia solani, Exserohilum turcicum (Northern corn leaf blight),Bipolaris maydis (Southern corn leaf blight), Ustilago maydis (cornsmut), Fusarium graminearum (Gibberella zeae), Fusarium verticilliodes{Gibberella moniliformis), F. proliferatum (G. fujikuroi var.intermedia), F. sub glutinous (G. subglutinans), Diplodia maydis,Sporisorium holci-sorghi, Colletotrichum graminicola, Setosphaeriaturcica, Aureobasidium zeae, Phytophthora infestans, Phytophthora sojae,Sclerotinia sclerotiorum, and fungal species.

Non-limiting examples of invertebrate pests can include cyst nematodesHeterodera spp. such as soybean cyst nematode Heterodera glycines, rootknot nematodes Meloidogyne spp., lance nematodes Hoplolaimus spp., stuntnematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchus spp.,lesion nematodes Pratylenchus spp., ring nematodes Criconema spp.,foliar nematodes Aphelenchus spp. or Aphelenchoides spp., cornrootworms, Lygus spp., aphids and similar sap-sucking insects such asphylloxera (Daktulosphaira vitifoliae), corn borers, cutworms,armyworms, leafhoppers, Japanese beetles, grasshoppers, and other pestcoleopterans, dipterans, and lepidopterans. Additional examples ofinvertebrate pests can include pests that can infest the root systems ofcrop plants, e.g., northern corn rootworm (Diabrotica barberi), southerncorn rootworm (Diabrotica undecimpunctata), Western corn rootworm(Diabrotica virgifera), corn root aphid (Anuraphis maidiradicis), blackcutworm (Agrotis ipsilon), glassy cutworm (Crymodes devastator), dingycutworm (Feltia ducens), claybacked cutworm (Agrotis gladiaria),wireworm (Melanotus spp., Aeolus mellillus), wheat wireworm (Aeolusmancus), sand wireworm (Horistonotus uhlerii), maize billbug(Sphenophorus maidis), timothy billbug (Sphenophorus zeae), bluegrassbillbug (Sphenophorus parvulus), southern corn billbug (Sphenophoruscallosus), white grubs (Phyllophaga spp.), seedcorn maggot (Deliaplatura), grape colaspis (Colaspis brunnea), seedcorn beetle(Stenolophus lecontei), and slender seedcorn beetle (Cliviniaimpressifrons), and parasitic nematodes.

A target gene of interest (e.g., for gene silencing) may include anycoding or non-coding sequence from any species (including, but notlimited to, eukaryotes such as fungi; plants, including monocots anddicots, such as crop plants, ornamental plants, and non-domesticated orwild plants; invertebrates such as arthropods, annelids, nematodes, andmollusks; and vertebrates such as amphibians, fish, birds, and mammals).Non-limiting examples of a non-coding sequence (e.g., that can beexpressed by a gene expression element such as a regulatory sequence)include, but not limited to, 5′ untranslated regions, promoters,enhancers, or other non-coding transcriptional regions, 3′ untranslatedregions, terminators, introns, microRNAs, microRNA precursor DNAsequences, small interfering RNAs, RNA components of ribosomes orribozymes, small nucleolar RNAs, and other non-coding RNAs. Non-limitingexamples of a gene of interest further include, but are not limited to,translatable (coding) sequence, such as genes encoding transcriptionfactors and genes encoding enzymes involved in the biosynthesis orcatabolism of molecules of interest (such as amino acids, fatty acidsand other lipids, sugars and other carbohydrates, biological polymers,and secondary metabolites including alkaloids, terpenoids, polyketides,non-ribosomal peptides, and secondary metabolites of mixed biosyntheticorigin).

The target gene (e.g., for gene silencing) may be an essential gene ofthe plant pest or plant pathogen. Essential genes can include genes thatmay be required for development of the pest or pathogen to a fertilereproductive adult. Essential genes can include genes that, whensilenced or suppressed, can result in the death of the organism (e.g.,as an adult or at any developmental stage, including gametes) or in theorganism's inability to successfully reproduce (e.g., sterility in amale or female parent or lethality to the zygote, embryo, or larva).Non-limiting examples of nematode essential genes include major spermprotein, RNA polymerase II, and chitin synthase. Additional soybean cystnematode essential genes are provided in U.S. Patent PublicationUS20070271630, incorporated by reference herein. The gene can be aDrosophila essential gene. The gene can be a fungal essential gene.

Target genes (e.g., from pests) can include invertebrate genes for majorsperm protein, alpha tubulin, beta tubulin, vacuolar ATPase,glyceraldehyde-3-phosphate dehydrogenase, PvNA polymerase TT, chitinsynthase, cytochromes, miRNAs, miRNA precursor molecules and miRNApromoters. Target genes (e.g., from pathogens) can include genes formiRNAs, miRNA precursor molecules, fungal tubulin, fungal vacuolarATPase, fungal chitin synthase, fungal MAP kinases, fungal Pad Tyr/Thrphosphatase, enzymes involved in nutrient transport (e.g., amino acidtransporters or sugar transporters), enzymes involved in fungal cellwall biosynthesis, cutinases, melanin biosynthetic enzymes,polygalacturonases, pectinases, pectin lyases, cellulases, proteases,genes that interact with plant avirulence genes, and genes involved ininvasion and replication of the pathogen in the infected plant.

Plants may be transformed (e.g., in the nucleus, an organelle, or both)with an expression cassette encoding, for example, a dsRNA, a siRNA or amiRNA. The dsRNA, siRNA, or miRNA can suppress (e.g., expression of) atleast one (e.g., at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10)target gene present in a plant pest. The dsRNA, siRNA, or miRNA cansuppress, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or moretarget genes of a plant pest. Suppression of a target gene present inthe plant pest can provide complete or nearly complete protection fromthe plant pest. “Complete protection” can mean that no (e.g.,substantial) damage can be caused to the plant by the plant pest.

The dsRNA, the siRNA or the miRNA may be designed for suppression of agene selected from the group consisting of: proteasome A-type subunitpeptide (Pas-4), ACT, SHR, EPIC2B and PnPMAI.

SEQ ID NO:114 corresponds to an open reading frame encoding anHeterodera glycines (SCN) specific proteasome A-type subunit peptidethat can be referred to herein as Pas-4. SEQ ID NO: 115 corresponds tonucleotides 552-699 of SEQ ID NO: 114. SEQ ID NO: 115 or SEQ ID NO: 114can be useful for dsRNA-mediated suppression of Pas-4. ACT can encodeβ-actin, which can be an essential cytoskeletal protein. SHR can encodeShrub (also known as Vps32 or Snf7), which can be an essential subunitof a protein complex involved in membrane remodeling for vesicletransport. EPIC2B can encode a Phytophthora infestans protein that caninteract with and/or inhibit a novel papain-like extracellular Cysprotease, for example, Phytophthora Inhibited Protease 1. The PnPMA genefrom Phytophthora parasitica can encode a plasma membrane H⁺ ATPase.

Resistance to Plant Pests

Resistance to pests in plants can be achieved by, for example,transgenic control. In-plant transgenic control of, for example, insectpests, can be achieved through, for example, plant expression of crystal(Cry) delta endotoxin genes and/or Vegetative Insecticidal Proteins(VIP) such as from Bacillus thuringiensis. Non-limiting examples of Crytoxins include, for example, the 60 main groups of “Cry” toxins (e.g.,Cry1-Cry59) and VIP toxins. Cry toxins can include subgroups of Crytoxins, for example, Cry 1a.

An expression cassette for use in transformation (e.g., into anorganelle) may be constructed using, for example, a Cry sequence. TheCry sequence can include, for example, the wild-type (e.g., native)nucleic acid sequence encoding at least one protein selected from thegroup consisting of: Cry1Ac, Cyt1Aa, Cry1Ab, Cry2Aa, Cry1I, Cry1C,Cry1D, Cry1E, Cry1Be, Cry1Fa and Vip3A. The Cry sequence can include,for example, a modified (e.g., truncated or fusion) nucleic acidsequence encoding at least one protein selected from the groupconsisting of: Cry1Ac, Cyt1Aa, Cry1Ab, Cry2Aa, Cry1I, Cry1C, Cry1D,Cry1E, Cry1Be, Cry1Fa and Vip3A. A modified such as a truncated nucleicacid sequence can encode a modified such as a truncated protein fragmentthat can retain insecticidal activity. The nucleic acid sequenceencoding the full-length, or modified (e.g., truncated) protein may becodon-optimized for the organelle of interest. The Cry protein can be aCyt1Aa protein (e.g., from Bacillus thuringiensis serovar israelensis;Gene ID: 5759908; SEQ ID NO:111).

Accessory proteins, for example, for a Cry protein, can be introducedinto a cell (e.g., into an organelle). An accessory protein can, forexample, increase expression, stability, and/or function of, forexample, a Cry protein. Non-limiting examples of accessory proteinsinclude 20 kDa accessory proteins (e.g., from Bacillus thuringiensisserovar israelensis) and 19 kDa accessory proteins (e.g., from Bacillusthuringiensis serovar israelensis). The accessory protein can be the 20kDa accessory protein from Bacillus thuringiensis serovar israelensis(pBt024; SEQ ID NO:112). The accessory protein can be the 19 kDaaccessory protein from Bacillus thuringiensis serovar israelensis,(pBt022; SEQ ID NO:113). Accessory proteins can be included in anexpression cassette as a polycistronic unit. Accessory proteins can beexpressed from separate expression cassettes.

Polynucleotides that encode proteins useful in conferring insectresistance to a plant may be included in an expression cassette as apolycistronic unit, or may be expressed from separate expressioncassettes. In some embodiments, these polynucleotides can encode thefollowing: (a) the Cyt1Aa protein from Bacillus thuringiensis serovarisraelensis (Gene ID: 5759908; SEQ ID NO:111); (b) the 20 kDa accessoryprotein from Bacillus thuringiensis serovar israelensis (pBt024; SEQ IDNO:112); and (c) the 19 kDa accessory protein from Bacillusthuringiensis serovar israelensis, (pBt022; SEQ ID NO:113).

Genome Modification

The disclosure provides compositions and methods that can be used for,for example, genome modification of a target sequence in the genome(e.g., a plastid or a mitochondrial genome) of an organism or cell(e.g., a plant or plant cell), for selecting the modified organism orcell, for gene editing, and for inserting a donor polynucleotide intothe genome of an organism or cell. The methods can employ apolynucleotide guided polypeptide system; e.g., a guidepolynucleotide/Cas protein system. The Cas protein can be guided by theguide polynucleotide to recognize a target polynucleic acid. The Casprotein can introduce a single strand or double strand break at aspecific target site into the genome of a cell. The guidepolynucleotide/Cas polypeptide system can provide for an effectivesystem for modifying target sites within the genome of a plant, plantcell or seed.

A variety of methods can be employed to further modify a target site tointroduce a donor polynucleotide of interest. The nucleotide sequence tobe edited (e.g., the nucleotide sequence of interest) can be locatedwithin or outside a target site that is recognized by a polynucleotideguided polypeptide.

Further provided are methods and compositions employing a polynucleotideguided polypeptide system for modification of multiple target siteswithin the genome of an organelle. Modification of multiple target siteswithin the genome of an organelle can facilitate the creation ofhomoplastic transformation events.

Polynucleotide Guided Polypeptide Systems

A polynucleotide-guided polypeptide can be a polypeptide that can bindto a target nucleic acid. A polynucleotide-guided polypeptide can be anuclease. A polynucleotide-guided polypeptide can be an endonuclease. Apolynucleotide-guided polypeptide can be a Cas protein. Apolynucleotide-guided polypeptide can be an Argonaut protein. Apolynucleotide guided polypeptide can form a complex with a guidepolynucleotide. A polynucleotide guided polypeptide can be directed to atarget nucleic acid by a guide polynucleotide. A polynucleotide guidedpolypeptide can complex with a guide polynucleotide to recognize atarget nucleic acid. A polynucleotide guided polypeptide can introduce asingle strand or double strand break at a specific target site (e.g.,the genome of a cell).

a. CRISPR Loci

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats)(also known as SPIDRs-SPacer Interspersed Direct Repeats) can constitutea family of DNA loci. CRISPR loci can consist of short and highlyconserved DNA repeats (e.g., 24 to 40 bp, repeated from 1 to 140times-also referred to as CRISPR-repeats). CRISPR DNA repeats can bepartially palindromic. The repeated sequences (e.g., usually specific toa species) can be interspaced by variable sequences of constant length(e.g., 20 to 58 by depending on the CRISPR locus.

CRISPR loci can occur in, for example, E. coli, Haloferax mediterranei,Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis. TheCRISPR loci can comprise short regularly spaced repeats (SRSRs). Therepeats can be short elements that can occur in clusters. The repeatscan be regularly spaced by variable sequences of constant length.

CRISPR systems can belong to different classes, with different repeatpatterns, sets of genes, and species ranges. The number of Cas genes ata given CRISPR locus can vary between species.

b. Cas Protein

A Cas protein can be a protein of a CRISPR/Cas system. A Cas protein canbe a Class 1 or a Class 2 Cas protein. A Cas protein can be a Type I,Type II, Type III, Type IV, Type V, or Type VI Cas protein.

“Cas gene” can refer to a gene that encodes a Cas protein. The terms Casprotein and Cas polypeptide can be used interchangeably herein. Cas genecan be coupled, associated or close to or in the vicinity of flankingCRISPR loci. The terms “Cas gene”, “CRISPR-associated (Cas) gene” can beused interchangeably herein.

A Cas protein can bind to a target nucleic acid. A Cas protein can be aCas nuclease. A Cas protein can be a Cas endonuclease. A Cas protein cancomplex with a guide polynucleotide. A Cas protein can be directed to atarget nucleic acid by a guide polynucleotide. A Cas protein can complexwith a guide polynucleotide to recognize a target nucleic acid. A Casprotein can introduce a single strand or double strand break at a targetnucleic acid sequence (e.g., DNA or RNA). A Cas protein can be enabledby the guide polynucleotide to recognize and introduce a single strandor double strand break at a specific target site into the genome of acell.

A Cas protein can comprise one or more domains. Non-limiting examples ofdomains include, guide nucleic acid recognition and/or binding domain,nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA bindingdomain, RNA binding domain, helicase domains, protein-proteininteraction domains, and dimerization domains. A guide nucleic acidrecognition and/or binding domain can interact with a guide nucleicacid. A nuclease domain can comprise catalytic activity for nucleic acidcleavage. A nuclease domain can lack catalytic activity to preventnucleic acid cleavage. A Cas protein can be a chimeric Cas protein thatis fused to other proteins or polypeptides. A Cas protein can be achimera of various Cas proteins, for example, comprising domains fromdifferent Cas proteins (e.g., homologues).

Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7,Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10,Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1(CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

A Cas protein may be from any suitable organism. Non-limiting examplesinclude Streptococcus pyogenes, Streptococcus thermophilus,Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei,Streptomyces pristinae spiralis, Streptomyces viridochromo genes,Streptomyces viridochromogenes, Streptosporangium roseum,Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacilluspseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum,Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina,Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonassp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa,Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum,Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichiashahii, and Francisella novicida. In some aspects, the organism can beStreptococcus pyogenes (S. pyogenes).

A Cas protein as used herein can be a wildtype or a modified form of aCas protein. A Cas protein can be an active variant, inactive variant,or fragment of a wild type or modified Cas protein. A Cas protein cancomprise an amino acid change such as a deletion, insertion,substitution, variant, mutation, fusion, chimera, or any combinationthereof relative to a wild-type version of the Cas protein. A Casprotein can be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity or sequence similarity to a wild type exemplaryCas protein (e.g., Cas9 from S. pyogenes). A Cas protein can be apolypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% sequence identity and/or sequence similarity to a wildtype exemplary Cas protein. Variants or fragments can comprise at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequencesimilarity to a wild type or modified Cas protein or a portion thereof.Variants or fragments can be targeted to a nucleic acid locus in complexwith a guide nucleic acid while lacking nucleic acid cleavage activity.

A Cas protein can comprise one or more nuclease domains, such as DNasedomains. For example, a Cas9 protein can comprise a RuvC-like nucleasedomain and/or an HNH-like nuclease domain. The RuvC and HNH domains caneach cut a different strand of double-stranded DNA to make adouble-stranded break in the DNA. A Cas protein can comprise only onenuclease domain (e.g., Cpf1 comprises RuvC domain but lacks HNH domain)

A Cas protein can comprise an amino acid sequence having at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequencesimilarity to a nuclease domain (e.g., RuvC domain, HNH domain) of awild-type Cas protein.

A Cas protein can be modified to optimize activity e.g., cleavage,regulation of gene expression. A Cas protein can be modified to increaseor decrease nucleic acid binding affinity, nucleic acid bindingspecificity, and/or enzymatic activity. Cas proteins can also bemodified to change any other activity or property of the protein, suchas stability. For example, one or more nuclease domains of the Casprotein can be modified, deleted, or inactivated, or a Cas protein canbe truncated to remove domains that are not essential for the functionof the protein or to optimize (e.g., enhance or reduce) the activity ofthe Cas protein.

A Cas protein can be a fusion protein. For example, a Cas protein can befused to a cleavage domain, an epigenetic modification domain, atranscriptional activation domain, or a transcriptional repressordomain. A Cas protein can also be fused to a heterologous polypeptideproviding increased or decreased stability. The fused domain orheterologous polypeptide can be located at the N-terminus, theC-terminus, or internally within the Cas protein.

A Cas protein can comprise a heterologous polypeptide for ease oftracking or purification, such as a fluorescent protein, a purificationtag, or an epitope tag. Examples of fluorescent proteins include greenfluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald,Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellowfluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP,ZsYellow1), blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite,mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g.eCFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescentproteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins(mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine,tdTomato), and any other suitable fluorescent protein. Examples of tagsinclude glutathione-S-transferase (GST), chitin binding protein (CBP),maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG,hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV,KT3, S, SI, T7, V5, VSV-G, histidine (His), biotin carboxyl carrierprotein (BCCP), and calmodulin.

A Cas protein can be provided in any form. For example, a Cas proteincan be provided in the form of a protein, such as a Cas protein alone orcomplexed with a guide nucleic acid. A Cas protein can be provided inthe form of a nucleic acid encoding the Cas protein, such as an RNA(e.g., messenger RNA (mRNA)) or DNA.

The nucleic acid encoding the Cas protein can be codon optimized forefficient translation into protein in a particular cell, organelles, ororganism.

Nucleic acids encoding Cas proteins can be stably integrated in thegenome of an organelle or a cell. Nucleic acids encoding Cas proteinscan be operably linked to a promoter active in the cell. Nucleic acidsencoding Cas proteins can be operably linked to a promoter in anexpression construct. Expression constructs can include any nucleic acidconstructs that can direct expression of a gene or other nucleic acidsequence of interest (e.g., a Cas gene). Expression constructs caninclude any nucleic acid constructs that can transfer such a nucleicacid sequence of interest to a target cell (e.g., into an organelle).

In some aspects, a Cas protein can be a Class 2 Cas protein. In someaspects, a Cas protein can be a type II Cas protein. In some aspects,the Cas protein can be a Cas9 protein, a modified version of a Cas9protein, or derived from a Cas9 protein.

Cas9 can refer to a polypeptide with at least about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequencesimilarity to a wild type exemplary Cas9 polypeptide (e.g., Cas9 from S.pyogenes). Cas9 can refer to a polypeptide with at most about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/orsequence similarity to a wild type exemplary Cas9 polypeptide (e.g.,from S. pyogenes). Cas9 can refer to the wildtype or a modified form ofthe Cas9 protein that can comprise an amino acid change such as adeletion, insertion, substitution, variant, mutation, fusion, chimera,or any combination thereof.

In one embodiment, the polynucleotide guided polypeptide gene can be aCas9 protein, such as but not limited to, Cas9 sequences listed in SEQID NOs: 462, 474, 489, 494, 499, 505, and 518 of WO2007/025097 andincorporated herein by reference. The Cas9 protein can unwind the DNAduplex in close proximity of the genomic target site. The Cas9 proteincan cleave for example both DNA strands upon recognition of a targetsequence by a guide polynucleic acid. In some aspects, the Cas9endonuclease can cleave only if the correct protospacer-adjacent motif(PAM) is approximately oriented at the 3′ end of the target sequence.Mutagenesis of Streptococcus pyogenes Cas9 catalytic domains can produce“nicking” enzymes (Cas9n) that can induce single-strand nicks ratherthan double-strand breaks.

In another embodiment, the polynucleotide guided polypeptide codingsequence can be modified to use codons preferred by the target organism,e.g., a plant, maize or soybean codon-optimized sequence encoding a Cas(e.g., Cas9) protein. In another embodiment, the sequence that encodes apolynucleotide guided polypeptide can be operably linked to one or moresequences encoding nuclear localization signals; e.g., to a SV40 nucleartargeting signal upstream of the Cas protein coding region and abipartite VirD2 nuclear localization signal downstream of the Casprotein coding region.

In another embodiment, the polynucleotide guided polypeptide may be anArgonaute protein such as Natronobacterium gregoryi Argonaute (“NgAgo”).The Argonaute protein can be a DNA-guided endonuclease. Argonauteproteins can bind a guide DNA such as a 5′-phosphorylatedsingle-stranded guide DNA (gDNA) of for example, 24 nucleotides.Argonaute proteins can create site-specific target nucleic acid (e.g.,DNA) breaks (e.g., double-stranded breaks) when loaded with the gDNA.The Argonaute protein—gDNA system may not require a protospacer-adjacentmotif (PAM) for recognition of a target nucleic acid.

In some aspects, the polynucleotide guided polypeptide can be a dead Casprotein. A Cas protein can be a dead Cas protein. A dead Cas protein canbe a protein that lacks nucleic acid cleavage activity.

A Cas protein can comprise a modified form of a wild type Cas protein.The modified form of the wild type Cas protein can comprise an aminoacid change (e.g., deletion, insertion, or substitution) that reducesthe nucleic acid-cleaving activity of the Cas protein. For example, themodified form of the Cas protein can have less than less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% of the nucleic acid-cleaving activity of the wild-type Cas protein(e.g., Cas9 from S. pyogenes). The modified form of Cas protein can haveno substantial nucleic acid-cleaving activity. When a Cas protein is amodified form that has no substantial nucleic acid-cleaving activity, itcan be referred to as enzymatically inactive and/or “dead” (abbreviatedby “d”). A dead Cas protein (e.g., dCas, dCas9) can bind to a targetpolynucleotide but may not cleave the target polynucleotide. In someaspects, a dead Cas protein can be a dead Cas9 protein.

Enzymatically inactive can refer to a polypeptide that can bind to anucleic acid sequence in a polynucleotide in a sequence-specific manner,but may not cleave a target polynucleotide. An enzymatically inactivesite-directed polypeptide can comprise an enzymatically inactive domain(e.g. nuclease domain). Enzymatically inactive can refer to no activity.Enzymatically inactive can refer to substantially no activity.Enzymatically inactive can refer to essentially no activity.Enzymatically inactive can refer to an activity less than 1%, less than2%, less than 3%, less than 4%, less than 5%, less than 6%, less than7%, less than 8%, less than 9%, or less than 10% activity compared to awild-type exemplary activity (e.g., nucleic acid cleaving activity,wild-type Cas9 activity).

One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Casprotein can be deleted or mutated so that they are no longer functionalor comprise reduced nuclease activity. For example, in a Cas proteincomprising at least two nuclease domains (e.g., Cas9), if one of thenuclease domains is deleted or mutated, the resulting Cas protein, knownas a nickase, can generate a single-strand break at a CRISPR RNA (crRNA)recognition sequence within a double-stranded DNA but not adouble-strand break. Such a nickase can cleave the complementary strandor the non-complementary strand, but may not cleave both. If all of thenuclease domains of a Cas protein (e.g., both RuvC and HNH nucleasedomains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein) aredeleted or mutated, the resulting Cas protein can have a reduced or noability to cleave both strands of a double-stranded DNA. An example of amutation that can convert a Cas9 protein into a nickase can be a D10A(aspartate to alanine at position 10 of Cas9) mutation in the RuvCdomain of Cas9 from S. pyogenes. H939A (histidine to alanine at aminoacid position 839) or H840A (histidine to alanine at amino acid position840) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9into a nickase. An example of a mutation that can convert a Cas9 proteininto a dead Cas9 is a D10A (aspartate to alanine at position 10 of Cas9)mutation in the RuvC domain and H939A (histidine to alanine at aminoacid position 839) or H840A (histidine to alanine at amino acid position840) in the HNH domain of Cas9 from S. pyogenes.

A dead Cas protein can comprise one or more mutations relative to awild-type version of the protein. The mutation can result in less than90%, less than 80%, less than 70%, less than 60%, less than 50%, lessthan 40%, less than 30%, less than 20%, less than 10%, less than 5%, orless than 1% of the nucleic acid-cleaving activity in one or more of theplurality of nucleic acid-cleaving domains of the wild-type Cas protein.The mutation can result in one or more of the plurality of nucleicacid-cleaving domains retaining the ability to cleave the complementarystrand of the target nucleic acid but reducing its ability to cleave thenon-complementary strand of the target nucleic acid. The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the non-complementary strand of thetarget nucleic acid but reducing its ability to cleave the complementarystrand of the target nucleic acid. The mutation can result in one ormore of the plurality of nucleic acid-cleaving domains to lack theability to cleave the complementary strand and the non-complementarystrand of the target nucleic acid. The residues to be mutated in anuclease domain can correspond to one or more catalytic residues of thenuclease. For example, residues in the wild type exemplary S. pyogenesCas9 polypeptide such as Asp10, His840, Asn854 and Asn856 can be mutatedto inactivate one or more of the plurality of nucleic acid-cleavingdomains (e.g., nuclease domains). The residues to be mutated in anuclease domain of a Cas protein can correspond to residues Asp10,His840, Asn854 and Asn856 in the wild type S. pyogenes Cas9 polypeptide,for example, as determined by sequence and/or structural alignment.

As non-limiting examples, residues D10, G12, G17, E762, H840, N854,N863, H982, H983, A984, D986, and/or A987 (or the correspondingmutations of any of the Cas proteins) can be mutated. For example, e.g.,D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A,and/or D986A. Mutations other than alanine substitutions can besuitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a polynucleotide guided polypeptide (e.g.,Cas9 protein) substantially lacking DNA cleavage activity (e.g., a deadCas9 protein).

In another embodiment, the polynucleotide guided polypeptide can be apolypeptide moiety (e.g., a chimeric polypeptide) that can form aprogrammable nucleoprotein molecular complex with a specificityconferring nucleic acid (SCNA). The programmable nucleoprotein molecularcomplex can assemble in-vivo, in a target cell, or in an organelle. Theprogrammable nucleoprotein molecular complex can interact with apredetermined target nucleic acid sequence. The programmablenucleoprotein molecular complex may comprise a polynucleotide moleculeencoding a chimeric polypeptide. The chimeric polypeptide can comprise afunctional domain that can modify a target nucleic acid site. Thefunctional domain can be devoid of a specific nucleic acid binding site.The chimeric polypeptide can comprise a linking domain that can interactwith a SCNA. The linking domain can be devoid of a specific targetnucleic acid binding site. A SCNA can comprise a nucleotide sequencecomplementary to a region of a target nucleic acid flanking the targetsite. A SCNA can comprise a recognition region that can specificallyattach to the linking domain of a chimeric polypeptide. Assembly of thechimeric polypeptide and the SCNA within the target cell can form afunctional nucleoprotein complex. The nucleoprotein complex canspecifically modify a target nucleic acid at the target site.

In another embodiment, the polynucleotide guided endonuclease gene canbe a full-length polynucleotide guided endonuclease (e.g., Casendonuclease, Cas9 endonuclease), or any functional fragment orfunctional variant thereof.

The terms “functional fragment”, “fragment that is functionallyequivalent” and “functionally equivalent fragment” can be usedinterchangeably herein. In the context of a sequence encoding apolynucleotide guided polypeptide, these terms can refer to a portion orsubsequence of the polynucleotide guided polypeptide sequence. Theportion or subsequence of the polynucleotide guided polypeptide sequencecan comprise the ability to create a single-strand or double-strandbreak.

The terms “functional variant”, “variant that is functionallyequivalent” and “functionally equivalent variant” can be usedinterchangeably herein. In the context of a polynucleotide guidedpolypeptide, these terms can refer to a variant of the polynucleotideguided polypeptide. The variant can comprise the ability to create asingle-strand or double-strand break. Fragments and variants can beobtained via methods such as site-directed mutagenesis and syntheticconstruction.

In one embodiment, the polynucleotide guided polypeptide coding sequencecan be a plant codon-optimized Streptococcus pyogenes Cas9 codingsequence. The codon optimized Cas9 sequence can recognize any genomicsequence, for example, of the form N(12-30)NGG.

In one embodiment, the polynucleotide guided polypeptide can beintroduced directly into a cell by any suitable method, for example, butnot limited to transient introduction methods, transfection and/ortopical application.

Compositions and methods of the disclosure can use endonucleases.Endonucleases can be enzymes that cleave the phosphodiester bond withina polynucleotide chain. Endonucleases can include restrictionendonucleases that cleave DNA at specific sites without damaging thebases. Restriction endonucleases can include Type I, Type II, Type III,and Type IV endonucleases, which can further include subtypes. In theType I and Type III systems, both the methylase and restrictionactivities can be contained in a single complex. Endonucleases can alsoinclude meganucleases, also known as homing endonucleases (HEases).Meganucleases can bind and cut at a specific recognition site, which canbe about 18 bp or more. Meganucleases can be classified into fourfamilies based on conserved sequence motifs. The meganuclease familiescan be LAGLIDADG, GIY-YIG, H—N—H, and His-Cys box families. These motifscan participate in the coordination of metal ions and hydrolysis ofphosphodiester bonds. HEases can have long recognition sites, and cantolerate sequence polymorphisms in their DNA substrates. The namingconvention for meganuclease can be similar to the convention for otherrestriction endonuclease.

Meganucleases can also be characterized by prefix F-, I-, or PI- forenzymes encoded by free-standing ORFs, introns, and inteins,respectively. One step in the recombination process can involvepolynucleotide cleavage at or near the recognition site. This cleavingactivity can be used to produce a double-strand break. In some examplesthe recombinase can be from the Integrase or Resolvase families.

Compositions and methods of the disclosure can use Transcriptionactivator-like effector nucleases (TALENs; TAL effector nucleases) canbe a class of sequence-specific nucleases. TALENs can be used to cleave(e.g., double-strand breaks) at specific target sequences (e.g., in thegenome of a plant or other organism). TAL effector nucleases can becreated by fusing a native or engineered transcription activator-like(TAL) effector, or functional part thereof, to the catalytic domain ofan endonuclease, such as, for example, Fokl. The unique, modular TALeffector DNA binding domain can allow for the design of proteins withpotentially any given DNA recognition specificity.

Compositions and methods of the disclosure can use zinc finger nucleases(ZFNs). ZFNs can be engineered cleavage (e.g., double-strand break)inducing agents comprised of a zinc finger DNA binding domain and adouble-strand-break-inducing agent domain. Recognition site specificitycan be conferred by the zinc finger domain, which can comprise two,three, or four zinc fingers, for example having a C2H2 structure. Zincfinger domains can be amenable for designing polypeptides whichspecifically bind a selected polynucleotide recognition sequence. ZFNscan consist of an engineered DNA-binding zinc finger domain linked to anon-specific endonuclease domain, for example, a nuclease domain from aType IIS endonuclease such as Fokl. Additional functionalities can befused to the zinc-finger binding domain, including transcriptionalactivator domains, transcription repressor domains, and methylases. Insome examples, dimerization of nuclease domain may be required forcleavage activity. Each zinc finger can recognize, for example, threeconsecutive base pairs in the target DNA. For example, a 3 finger domainrecognized a sequence of 9 contiguous nucleotides, with a dimerizationrequirement of the nuclease, two sets of zinc finger triplets can beused to bind an 18 nucleotide recognition sequence.

c. Guide Polynucleic Acid

Bacteria and archaea can have evolved adaptive immune defenses termedclustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) systems that can use short RNA todirect degradation of foreign nucleic acids. The type II CRISPR/Cassystem from bacteria can employ a crRNA and tracrRNA to guide the Caspolypeptide to a nucleic acid target. The crRNA (CRISPR RNA) can containthe region complementary to one strand of the double strand DNA target.The crRNA can base pair with the tracrRNA (trans-activating CRISPR RNA)to form a RNA duplex that can direct the Cas polypeptide to recognizeand optionally cleave the DNA target.

As used herein, the term “guide polynucleotide”, can refer to apolynucleotide sequence that can form a complex with a polynucleotideguided polypeptide (e.g., a Cas protein). The guide polynucleotide candirect the polynucleotide guided polypeptide to recognize and optionallycleave (or nick) a DNA target site. The terms “guide polynucleotide” and“guide polynucleic acid” can be used interchangeably herein. The guidepolynucleotide can be comprised of a single molecule (unimolecular) ortwo molecules (bimolecular). The guide polynucleotide sequence can be aRNA sequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence).

Optionally, the guide polynucleotide can comprise at least onenucleotide, phosphodiester bond or linkage modification such as, but notlimited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine,2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA, phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule,or 5′ to 3′ covalent linkage resulting in circularization. A guidepolynucleotide that solely comprises ribonucleic acids can also bereferred to as a “guide RNA” (gRNA). In some embodiments, the guidepolynucleic acid can be a guide RNA.

As used herein, the term “single guide RNA” (sgRNA) can refer to asynthetic fusion of two RNA molecules, for example, a crRNA (CRISPR RNA)comprising a variable targeting domain, and a tracrRNA. In oneembodiment, the guide RNA can comprise a variable targeting domain of 12to 30 nucleotide sequences and a RNA fragment that can interact with aCas protein.

As used herein, “crRNA” can refer to a nucleic acid with at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identityand/or sequence similarity to a wild type exemplary crRNA (e.g., a crRNAfrom S. pyogenes). crRNA can refer to a nucleic acid with at most about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identityand/or sequence similarity to a wild type exemplary crRNA (e.g., a crRNAfrom S. pyogenes). crRNA can refer to a modified form of a crRNA thatcan comprise a nucleotide change such as a deletion, insertion, orsubstitution, variant, mutation, or chimera. A crRNA can be a nucleicacid having at least about 60% identical to a wild type exemplary crRNA(e.g., a crRNA from S. pyogenes) sequence over a stretch of at least 6contiguous nucleotides. For example, a crRNA sequence can be at leastabout 60% identical, at least about 65% identical, at least about 70%identical, at least about 75% identical, at least about 80% identical,at least about 85% identical, at least about 90% identical, at leastabout 95% identical, at least about 98% identical, at least about 99%identical, or 100% identical, to a wild type exemplary crRNA sequence(e.g., a crRNA from S. pyogenes) over a stretch of at least 6 contiguousnucleotides

As used herein, “tracrRNA” can refer to a nucleic acid with at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequenceidentity and/or sequence similarity to a wild type exemplary tracrRNAsequence (e.g., a tracrRNA from S. pyogenes). tracrRNA can refer to anucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% sequence identity and/or sequence similarity to a wildtype exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).tracrRNA can refer to a modified form of a tracrRNA that can comprise anucleotide change such as a deletion, insertion, or substitution,variant, mutation, or chimera. A tracrRNA can refer to a nucleic acidthat can be at least about 60% identical to a wild type exemplarytracrRNA (e.g., a tracrRNA from S. pyogenes) sequence over a stretch ofat least 6 contiguous nucleotides. For example, a tracrRNA sequence canbe at least about 60% identical, at least about 65% identical, at leastabout 70% identical, at least about 75% identical, at least about 80%identical, at least about 85% identical, at least about 90% identical,at least about 95% identical, at least about 98% identical, at leastabout 99% identical, or 100% identical, to a wild type exemplarytracrRNA (e.g., a tracrRNA from S. pyogenes) sequence over a stretch ofat least 6 contiguous nucleotides.

A guide polynucleotide can be bimolecular (i.e., two molecules; alsoreferred to as “double molecule”, “dual” or “duplex” guidepolynucleotide) comprising, for example, a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that iscomplementary to a nucleotide sequence in a target polynucleic acid(e.g., target DNA) and a second nucleotide sequence domain (referred toas Cas endonuclease recognition domain or CER domain) that interactswith a Cas polypeptide. The VT domain can refer to the spacer region ofa guide polynucleic acid. The VT domain can comprise a spacer region ofa guide polynucleic acid. The spacer region can interact with aprotospacer region of a target nucleic acid in a sequence-specificmanner via hybridization (e.g., base pairing). The CER domain of thebimolecular guide polynucleotide can comprise two separate moleculesthat can be hybridized along a region of complementarity to form, forexample, a duplex or a partial duplex. The two separate molecules can beRNA, DNA, and/or RNA-DNA-combination sequences. In some embodiments, thefirst molecule of the duplex guide polynucleotide comprising a VT domainlinked to a CER domaincan bereferred to as “crDNA” (when composed of acontiguous stretch of DNA nucleotides) or “crRNA” (when composed of acontiguous stretch of RNA nucleotides), or “crDNA-RNA” (when composed ofa combination of DNA and RNA nucleotides). The crNucleotide can comprisea fragment of the crRNA naturally occurring in bacteria and archaea. Inone embodiment, the size of the fragment of the crRNA naturallyoccurring in bacteria and archaea that can be present in a crNucleotidedisclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.In some embodiments, the second molecule of the duplex guidepolynucleotide comprising a CER domain can bereferred to as “tracrRNA”(when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA”(when composed of a contiguous stretch of DNA nucleotides) or“tracrDNA-RNA” (when composed of a combination of DNA and RNAnucleotides. In one embodiment, the RNA that guides the RNA/Cas9polypeptide complex, can be a duplexed RNA comprising a duplexcrRNA-tracrRNA.

Complementarity between a guide polynucleic acid (e.g., the VT domain,spacer region) and a target polynucleic acid (e.g., protospacer) can beperfect, substantial, or sufficient. Perfect complementarity between twonucleic acids can mean that the two nucleic acids can form a duplex inwhich every base in the duplex can be bonded to a complementary base byWatson-Crick pairing. Substantial or sufficient complementary can meanthat a sequence in one strand may not be completely and/or perfectlycomplementary to a sequence in an opposing strand, but that sufficientbonding occurs between bases on the two strands to form a stable hybridcomplex in a set of hybridization conditions (e.g., salt concentrationand temperature).

A guide polynucleotide can also be a single molecule (i.e.,unimolecular), comprising a first nucleotide sequence domain (referredto as Variable Targeting domain or VT domain) that can be complementaryto a nucleotide sequence in a target polynucleic acid (e.g., target DNA)and a second nucleotide domain (referred to as Cas endonucleaserecognition domain or CER domain) that interacts with a Cas polypeptide.For a single molecule guide polynucleotide, the CER domain can be formedfrom a contiguous stretch of nucleotides that can be RNA, DNA, and/orRNA-DNA-combination sequence. The VT domain and/or the CER domain of asingle guide polynucleotide can comprise a RNA sequence, a DNA sequence,or a RNA-DNA-combination sequence. In some embodiments, the single guidepolynucleotide comprises a crNucleotide (comprising a VT domain linkedto a CER domain) linked to a tracrNucleotide (comprising a CER domain),wherein the linkage can be a nucleotide sequence comprising a RNAsequence, a DNA sequence, or a RNA-DNA combination sequence. The singleguide polynucleotide being comprised of sequences from the crNucleotideand tracrNucleotide may be referred to as “single guide RNA” (sgRNA;when composed of a contiguous stretch of RNA nucleotides) or “singleguide DNA” (sgDNA; when composed of a contiguous stretch of DNAnucleotides) or “single guide RNA-DNA” (sgDNA-RNA; when composed of acombination of DNA and RNA nucleotides). In one embodiment of thedisclosure, the single guide RNA (sgRNA) comprises a crRNA or crRNAfragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cassystem that can form a complex with a type II Cas polypeptide, whereinsaid guide RNA/Cas polypeptide complex can direct the Cas polypeptide toa plant genomic target site, enabling the Cas polypeptide to introduce adouble strand break into the genomic target site.

The term “variable targeting domain” or “VT domain” can be usedinterchangeably herein and can refer to a nucleotide sequence that canbe present in the guide polynucleotide. VT domain can be complementaryto one strand of a double stranded DNA target site. The percentcomplementation between the first nucleotide sequence domain (VT domain)and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%. The variable target domain can be at least 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length. In some embodiments, the variable target domaincan comprise at least 17 nucleotides that are complementary to at least17 nucleotides of a target polynucleic acid. In some embodiments, thevariable targeting domain can comprise a contiguous stretch ofnucleotides that are complementary to the target polynucleic acid. Insome embodiments, the nucleotides of the guide polynucleic acid that arecomplementary to the target polynucleic acid can be non-contiguous. Insome embodiments, the variable targeting domain can comprise acontiguous stretch of 12 to 30 nucleotides. The variable targetingdomain can be composed of a DNA sequence, a RNA sequence, a modified DNAsequence, a modified RNA sequence, or any combination thereof.

A target polynucleotide can be identified by identifying a protospaceradjacent motif (PAM) within a region of interest and selecting a regionof a desired size upstream or downstream of the PAM as the protospacer.A corresponding spacer sequence can be designed by determining thecomplementary sequence of the protospacer region.

The term “Cas endonuclease recognition domain” or “CER domain” of aguide polynucleotide can be used interchangeably herein and can refer toa nucleotide sequence (such as a second nucleotide sequence domain of aguide polynucleotide), that interacts with a Cas polypeptide. The CERdomain can be composed of a DNA sequence, a RNA sequence, a modified DNAsequence, a modified RNA sequence (see for example modificationsdescribed herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotideof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA combination sequence. In one embodiment, thenucleotide sequence linking the crNucleotide and the tracrNucleotide ofa single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 nucleotides in length. In another embodiment, the nucleotidesequence linking the crNucleotide and the tracrNucleotide of a singleguide polynucleotide can comprise a tetranucleotide loop sequence, suchas, but not limiting to a GAAA tetranucleotide loop sequence. Nucleotidesequence modification of the guide polynucleotide, VT domain and/or CERdomain can be selected from, but not limited to, the group consisting ofa 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence, a stabilitycontrol sequence, a sequence that forms a dsRNA duplex, a modificationor sequence that targets the guide polynucleotide to a subcellularlocation, a modification or sequence that provides for tracking, amodification or sequence that provides a binding site for proteins, aLocked Nucleic Acid (LNA), a 5-methyl-2′-deoxycytodine (5mdC), a2,6-Diaminopurine nucleotide, a 2′-Fluoroadenosine nucleotide, a2′-Fluorouridine nucleotide; a 2′-O-Methyl RNA nucleotide, aphosphorothioate (PS) bond, linkage to a cholesterol molecule, linkageto a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5′to 3′ covalent linkage, or any combination thereof. These modificationscan result in at least one additional beneficial feature, wherein theadditional beneficial feature can be selected from the group consistingof: modified or regulated stability, subcellular targeting, tracking, afluorescent label, a binding site for a protein or protein complex,modified binding affinity to complementary target sequence, modifiedresistance to cellular degradation, and increased cellular permeability.

In one embodiment, the guide RNA and Cas polypeptide can form a complexthat can enable the Cas polypeptide to introduce a single strand ordouble strand break at a DNA target site.

In one embodiment, the variable target domain can be 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides inlength.

In one embodiment, the guide RNA can comprise a crRNA (or crRNAfragment) and a tracrRNA (or tracrRNA fragment) of the type IICRISPR/Cas system that can form a complex with a type II Caspolypeptide. The guide RNA/Cas polypeptide complex can direct the Caspolypeptide to a target nucleic acid site (e.g., DNA target). The Caspolypeptide can introduce a double strand break into the DNA targetsite.

In one embodiment the guide polynucleic acid can be introduced into acell directly using any suitable method such as, but not limited to,particle bombardment or topical applications.

In another embodiment the guide polynucleic acid can be introducedindirectly by introducing a recombinant DNA molecule comprising apolynucleotide encoding the guide polynucleic acid operably linked to anuclear or organellar promoter that can transcribe the polynucleotide insaid nucleus or organelle, respectively.

In some embodiments, the guide polynucleic acid can be introduced into aplant cell via particle bombardment or Agrobacterium transformation of arecombinant DNA construct comprising a polynucleotide encoding the guidepolynucleic acid operably linked to a promoter functional in a plant;e.g., a plant U6 polymerase III promoter, a CaMV 35S polymerase IIpromoter.

In one embodiment, the guide polynucleic acid can be a duplexed RNAcomprising a duplex crRNA-tracrRNA. A single guide polynucleic acid(e.g., single guide RNA) can require one expression cassette to expressthe single guide RNA. A duplexed crRNA-tracrRNA can require one or moreexpression cassette needs to express the duplexed crRNA-tracrRNA.

A plurality of polynucleic acids can be multiplexed to target multipletarget nucleic acids. For example, 2, 3, 4, 5, 6, 7, 9, 10, or more than10 target nucleic acids can be targeted simultaneously or iteratively.Multiplexing can be used, as non-limiting examples, to generate largegenomic deletions, modify multiple different sequences at once, and/orin conjunction with dual-nickases to target a gene. In some examples,more than one CRISPR/Cas system can be delivered to target two or morenucleic acid sequence targets. Homologous Cas proteins can be used formultiplexing applications.

Target Sites for Genome Modification

The terms “target site”, “target sequence”, “target polynucleotide”,“target polynucleic acid”, “target locus”, “genomic target site”,“genomic target sequence”, and “genomic target locus” can be usedinterchangeably herein. Target polynucleic acid can refer to apolynucleotide sequence in the genome (e.g., plastid or mitochondrialgenome) of, for example, a plant cell. Target polynucleic acid can referto the site (e.g., in a genome) recognized by a guide polynucleic acid.Target polynucleic acid can refer to the site (e.g., in a genome) atwhich a single-strand or double-strand break can be induced (e.g., by aCas polypeptide). The target site can be an endogenous site in thegenome. The target site can be heterologous to the organism and therebynot be naturally occurring in the genome. Target site can be found in aheterologous genomic location compared to where it occurs in nature. Asused herein, terms “endogenous target sequence” and “native targetsequence” can be used interchangeably herein and can refer to a targetsequence that can be endogenous or native to the genome of the organism.Endogenous target sequence can occur at the endogenous or nativeposition of that target sequence in the genome of the organism.

A target polynucleic acid can be DNA, RNA, or both. In some embodiments,the target polynucleic acid can be DNA (e.g., target DNA). In someembodiments, the target polynucleic acid can be genomic DNA. In someembodiments, the target polynucleic acid can be nuclear genomic DNA. Insome embodiments, the target polynucleic acid can be organelle genomicDNA. In some embodiments, the target polynucleic acid can be nucleargenomic DNA and organelle genomic DNA.

The terms “artificial target site” and “artificial target sequence” canbe used interchangeably herein and can refer to a target sequence thathas been introduced into the genome of a plant. Such an artificialtarget sequence can be identical in sequence to an endogenous or nativetarget sequence in the genome of an organism but may be located in adifferent position (i.e., a non-endogenous or non-native position) inthe genome of the organism.

An “altered target site”, “altered target sequence”, “modified targetsite”, “modified target sequence” can be used interchangeably herein andcan refer to a target sequence as disclosed herein that can comprise atleast one alteration when compared to the non-altered target sequence.Such “alterations” can include, for example: (i) replacement of at leastone nucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Methods for modifying an organellar genomic target site are disclosedherein.

In one embodiment, a method for modifying a target site in the genome ofan organelle can comprise introducing a guide polynucleic acid (e.g,guide RNA, single guide RNA) into a plant cell. The plant cell cancomprise a polynucleotide guided polypeptide (e.g., a Cas polypeptide).The guide polynucleic acid and polynucleotide guided polypeptide canform a complex that can direct the polynucleotide guided polypeptide tointroduce a single strand or double strand break at the target site.

Also provided is a method for modifying a target site in the genome ofan organelle. The method can comprise introducing a guide polynucleicacid and a polynucleotide guided polypeptide (e.g., a Cas polypeptide)into the organelle. The guide polynucleic acid and polynucleotide guidedpolypeptide can form a complex. The complex can direct thepolynucleotide guided polypeptide to introduce a single strand or doublestrand break at the target site in the genome of the organelle.

Further provided is a method for modifying a target site in the genomeof an organelle. The method can comprise introducing a guide polynucleicacid and a donor polynucleotide (e.g. donor DNA) into an organelle. Theorganelle can comprise a polynucleotide guided polypeptide (e.g., a Caspolypeptide). The guide polynucleic acid and polynucleotide guidedpolypeptide can form a complex that can direct the polynucleotide guidedpolypeptide to introduce a single strand or double strand break at thetarget site. The donor polynucleotide can be inserted into the site ofcleavage in the genome.

Further provided is a method for modifying a target site in the genomeof an organelle. The method can comprise: a) introducing into anorganelle a guide polynucleic acid comprising a variable targetingdomain and a polynucleotide guided polypeptide (e.g., a Caspolypeptide), wherein said guide polynucleic acid and saidpolynucleotide guided polypeptide can form a complex that can enable thepolynucleotide guided polypeptide to introduce a single strand or doublestrand break at said target site; and, b) identifying at least oneorganelle that has a modification at said target site, wherein themodification includes at least one deletion or substitution of one ormore nucleotides in said target site.

Further provided, a method for modifying a target polynucleic acid(e.g., target DNA) sequence in the genome of an organelle, the methodcomprising: a)introducing into an organelle a first recombinant DNAconstruct that can express a guide polynucleic acid and a secondrecombinant DNA construct that can express a polynucleotide guidedpolypeptide (e.g., a Cas polypeptide), wherein said guide polynucleicacid and said polynucleotide guided polypeptide can form a complex thatcan enable the polynucleotide guided polypeptide to introduce a singlestrand or double strand break at said target site; and, b) identifyingat least one organelle that has a modification at said target site,wherein the modification includes at least one deletion or substitutionof one or more nucleotides in said target site.

The length of the target site can vary and includes, for example, targetsites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. The targetsite can be palindromic, that is, the sequence on one strand reads thesame in the opposite direction on the complementary strand. Thenick/cleavage site can be within the target sequence. The nick/cleavagesite can be outside of the target sequence. In another variation, thecleavage could occur at nucleotide positions immediately opposite eachother to produce a blunt end cut or, in other cases, the incisions couldbe staggered to produce single-stranded overhangs, also called “stickyends”, which can be either 5′ overhangs, or 3′ overhangs.

The target nucleic acid sequence can be 5′ or 3′ of the PAM. The targetnucleic acid sequence can be, for example, 16, 17, 18, 19, 20, 21, 22,or 23 bases immediately 5′ of the first nucleotide of the PAM. Thetarget nucleic acid sequence can be, for example, 16, 17, 18, 19, 20,21, 22, or 23 bases immediately 3′ of the last nucleotide of the PAM.The target nucleic acid sequence can be 20 bases immediately 5′ of thefirst nucleotide of the PAM. The target nucleic acid sequence can be 20bases immediately 3′ of the last nucleotide of the PAM.

Site-specific cleavage of a target nucleic acid by a polynucleotideguided polypeptide (e.g., Cas protein) can occur at locations determinedby base-pairing complementarity between the guide nucleic acid and thetarget nucleic acid. Site-specific cleavage of a target nucleic acid bya polynucleotide guided polypeptide (e.g., Cas protein) can occur atlocations determined by the protospacer adjacent motif (PAM). Forexample, the cleavage site of Cas (e.g., Cas9) can be about 1 to about25, or about 2 to about 5, or about 19 to about 23 base pairs (e.g., 3base pairs) upstream or downstream of the PAM sequence. In someembodiments, the cleavage site of Cas (e.g., Cas9) can be 3 base pairsupstream of the PAM sequence. In some embodiments, the cleavage site ofCas (e.g., Cpf1) can be 19 bases on the (+) strand and 23 base on the(−) strand, producing a 5′ overhang 5 nt in length. In some cases, thecleavage can produce blunt ends. In some cases, the cleavage can producestaggered or sticky ends with 5′ overhangs. In some cases, the cleavagecan produce staggered or sticky ends with 3′ overhangs.

Different organisms can comprise different PAM sequences. Different Casproteins can recognize different PAM sequences. For example, in S.pyogenes, the PAM can be a sequence in the target nucleic acid thatcomprises the sequence 5′-XRR-3′, where R can be either A or G, where Xcan be any nucleotide and X can be immediately 3′ of the target nucleicacid sequence targeted by the spacer sequence. The PAM sequence of S.pyogenes Cas9 (SpyCas9) can be 5′-XGG-3′, where X can be any DNAnucleotide and can be immediately 3′ of the CRISPR recognition sequenceof the non-complementary strand of the target DNA. The PAM of Cpf1 canbe 5′-TTX-3′, where X can be any DNA nucleotide and can be immediately5′ of the CRISPR recognition sequence.

Active variants of genomic target sites can also be used. Such activevariants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the giventarget site. The active variants can retain biological activity. Theactive variants can be recognized by a polynucleotide guided polypeptide(e.g., Cas protein). The active variants can be cleaved by apolynucleotide guided polypeptide (e.g., Cas protein). Assays can beused to measure the double-strand break of a target site by anendonuclease. Assays can measure the overall activity and/or specificityof an endonuclease on DNA substrates containing recognition sites (e.g.,target sites, active variants).

Methods for Integrating a Donor Polynucleotide

The disclosure provides methods to obtain an organelle comprising adonor polynucleotide. Such methods can employ homologous recombinationto provide integration of the polynucleotide at the target site. Apolynucleotide of interest can be provided to the organelle in a donorDNA molecule.

A donor polynucleotide can be a nucleic acid sequence (e.g., DNA, RNA,or both) that can be integrated into a target nucleic acid, for example,the genome of an organelle. The donor polynucleotide can be insertedinto a genome e.g., at a cleavage site of a polynucleotide guidedpolypeptide. The donor polynucleotide can be inserted into a genome byhomologous recombination. In some embodiments, the donor polynucleotidecan comprise DNA and can be referred to as donor DNA.

A donor polynucleotide of any suitable size can be integrated into agenome. In some embodiments, the donor polynucleotide integrated into agenome can be less than 3, about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300,350, 400, 450, 500 or more than 500 kilobases (kb) in length. In someembodiments, the donor polynucleotide integrated into a genome can be atleast about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450,500 or more than 500 (kb) in length. In some embodiments, the donorpolynucleotide integrated into a genome can be up to about 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more than 500 (kb) inlength.

A donor polynucleotide can comprise a polynucleotide of interest, apolynucleotide modification template, a heterologous expressioncassette, or both. A donor polynucleotide (e.g. donor DNA) can beflanked by a first and a second region of homology. The polynucleotidemodification template can be, for example, a single nucleotide change tocreate a different allele in the organelle genome. The first and secondregions of homology of the donor polynucleotide (e.g. donor DNA) canshare homology to a first and a second genomic region, respectively,present in or flanking the target site (e.g., of the organellar genome).

“Homology” can mean DNA sequences that are similar. Homology can mean,for example, nucleic acid sequences with about: 50%, 55%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity. Forexample, a “region of homology to a genomic region” can be a region ofDNA that has a similar sequence to a given “genomic region” in theorganellar genome. A region of homology can be of any length that can besufficient to promote homologous recombination at the cleaved targetsite. For example, the region of homology can comprise at least 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600,2700, 2800, 2900, 3000, 3100 or more bases in length such that theregion of homology has sufficient homology to undergo homologousrecombination with the corresponding genomic region. “Sufficienthomology” can indicate that two polynucleotide sequences can havesufficient structural similarity to act as substrates for a homologousrecombination reaction.

The donor polynucleotide (e.g., donor DNA) may comprise an expressioncassette (e.g., encoding a heterologous polynucleotide of interest). Thedonor polynucleotide may comprise multiple expression cassettes. Theexpression cassette may be a polycistronic expression cassette; e.g.,where multiple protein-coding regions, functional RNAs, or a combinationof both, are expressed under control of a single promoter.

A “donor RNA” can be a corresponding RNA molecule that comprises, forexample, the same nucleic acid sequence as a donor DNA; i.e., withuridylate (“U”) in place of deoxythymidylate (“T”). A “donorpolynucleotide” may be either a donor DNA or a donor RNA, or acombination of DNA and RNA. The donor polynucleotide may be eithersingle-stranded or double-stranded.

An alternative method for modification of an organellar genome can bethe replacement of part or all of the organelle DNA with a “replacementDNA”. Endogenous organellar DNA can be reduced or eliminated by use ofsite-specific endonucleases such as polynucleotide guided polypeptides(e.g., Cas polypeptide, Cas9 polypeptide). At the same time orsubsequently, a replacement DNA may be introduced. The term “replacementDNA” can refer to fragments of organellar DNA or complete organellar DNAthat can convey a new genotype and corresponding trait(s) whentransformed into the organelle. The terms “replacement DNA” and“replacement organellar DNA” can be used interchangeably herein. In thecase of organellar DNA fragments, they can be integrated into theremaining endogenous organellar DNA by homologous recombination. In thecase of complete organellar DNA replacement, the replacement DNA can beisolated from cultivars, lines, sub species and other species whichpossess DNA compositions distinct from the endogenous organellar DNA ofrecipient cells. In some embodiments, the replacement DNA can comprise aDNA element functioning as a DNA replication origin in the recipientorganelles.

A sequence functional as an origin of replication can be included withthe compositions (e.g., polynucleotides, constructs, cassettes) of thedisclosure. Such sequences can include origin of replication for anorganelle. The origin of replication sequence can be a plastid origin ofreplication (e.g., plastid rRNA intergenic region) sequence. The originof replication sequence can be a mitochondrial origin of replicationsequence.

As used herein, a “genomic region” can refer to a segment of achromosome in the genome of, for example, an organelle. Genomic regioncan be present on either side of the target site. Genomic region cancomprise a portion of the target site. The genomic region can compriseat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases. Thegenomic region can comprise sufficient homology to undergo homologousrecombination with the corresponding region of homology.

Donor polynucleotides, polynucleotides of interest and/or traits can bestacked together in a complex trait locus. The guidepolynucleotide/polypeptide system can be used to generate double strandbreaks and for stacking traits in a complex trait locus.

Two or more polynucleotides encoding RNA and/or proteins can be includedin a cassette as a polycistronic unit. Polynucleotides encoding RNA canbe expressed from separate cassettes.

In one embodiment, the guide polynucleotide/polypeptide system can beused for introducing one or more donor polynucleotides or one or moretraits of interest into one or more target sites by providing one ormore guide polynucleotides, one or more polynucleotide guidedpolypeptides (e.g., Cas polypeptides), and optionally one or more donorpolynucleotides (e.g. donor DNA) to a plant cell. An organism can beproduced from that cell that comprises an alteration at said one or moretarget sites of the organellar DNA, wherein the alteration can beselected from the group consisting of (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, and (iv) any combination of(i)-(iii).

The structural similarity between a given genomic region and thecorresponding region of homology flanking the donor polynucleotide (e.g.donor DNA) can be any degree of sequence identity that allows forhomologous recombination to occur. For example, the amount of homologyor sequence identity shared by the “region of homology” flanking thedonor polynucleotide (e.g. donor DNA) and the “genomic region” of theplant genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity, such that the sequencesundergo homologous recombination

The region of homology flanking the donor polynucleotide (e.g. donorDNA) can have homology to any sequence flanking the target site. Whilein some embodiments, the regions of homology share significant sequencehomology to the genomic sequence immediately flanking the target site,the regions of homology can be designed to have sufficient homology toregions that may be further 5′ or 3′ to the target site. In still otherembodiments, the regions of homology can also have homology with afragment of the target site along with downstream genomic regions. Inone embodiment, the first region of homology further comprises a firstfragment of the target site and the second region of homology comprisesa second fragment of the target site, wherein the first and secondfragments are dissimilar.

As used herein, “homologous recombination” can refer to the exchange ofDNA fragments between two DNA molecules at the sites of homology. Thefrequency of homologous recombination can be influenced by a number offactors. The length of the region of homology can affect the frequencyof homologous recombination events, for example, the longer the regionof homology, the greater the frequency. The length of the homologyregion needed to observe homologous recombination may vary amongspecies.

Intermolecular recombination can occur in plastids, for example,transplastomic plants can arise through site-specific integration offoreign sequences by homologous recombination with the flanking sequenceon the transformation vector.

The generation of novel plastome genotypes by transformation can rely onintegration of foreign sequence by intermolecular homologousrecombination (HR). Mechanistically similar to gene conversion, HR andrepair pathways can participate in the subsequent events that yieldhomoplasmic transplastomic cells and eventually stable transplastomicplants. Intra- or intermolecular recombination between repeatedsequences, both in wild-type plastomes, can generate, for example,inversions when repeats are palindromic or deletions when direct. Therole of HR proteins in damage repair may be compromised, for example,when foreign DNA is introduced, and through associated tissue cultureand selective pressure, as these manipulations can place additionalstress on recombination machinery leading to unintended events.

Among the DNA repair and recombination genes identified in the nucleargenomes of Oryza and Arabidopsis, about 19 and 17%, respectively, can betargeted to plastids.

Plastid-localized RecA (e.g., from P. sativum) can comprise DNA strandtransfer activity. RecA can be implicated in recombination-mediatedrepair of damaged ptDNA. Reduced RecA1 (AT1G79050) activity can lead toa destabilization and reduction in ptDNA. The reduction in plastome copynumber in mutant lines relative to wild type can suggest that RecA1 mayparticipate in recombination-mediated replication.

Methods of the disclosure can use any suitable plastid enzymes forhomologous DNA recombination pathway. The predominance of homologousrecombination in plastids can result from suppression of illegitimaterecombination by plastid-localized members of the whirly family ofsingle-stranded DNA binding proteins. HR activity in a cell can beoptimized by increasing HR pathway members.

To achieve efficient foreign sequence integration by homologousrecombination endogenous plastome sequences can be used to targetinsertions. A positive correlation can be present between the rate ofrecombination and the length and/or degree of sequence homology.

The minimum flanking sequence length for plastid transformation can beas little as 400 bp on either side of the expression cassette and can besufficient to obtain transformation at a reasonable frequency. Targetingsequences can extend from 1 to 1.5 kb on either size of the expressioncassette.

Non-homologous end-joining (NHEJ) can be a major DNA repair pathway inthe eukaryotic nucleus. NHEJ can also be active in bacteria and in plantmitochondria. In some cases, NHEJ may not occur in angiosperm plastids.NHEJ products can be produced in Arabidopsis. In some cases, repair ofDSBs by NHEJ following I-CreII activity can be detected at lowfrequency. NHEJ repair events can represent 17% of the rearrangedproducts in Whirly knockout lines. NHEJ can occur in plastids. NHEJ canbe a quantitatively minor pathway.

The methods of the disclosure can use homology-directed repair (HDR) orNHEJ. In some embodiments, HDR can be used. In some embodiments, theefficiency of HDR can be increased by, for example, increasingexpression of proteins and enzymes involved in HDR. In some embodiments,the efficiency of NHEJ can be reduced, by for example, targeting genesand/or proteins (e.g., DNA ligase) involved in NHEJ.

In some embodiments, the efficiency of the disclosed methods for genomeengineering or modification can be about 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

In one embodiment provided herein, the method can comprise contacting anorganelle of a plant cell with the donor polynucleotide (e.g. donorDNA), the guide polynucleic acid and the polynucleotide guidedpolypeptide. At least one one single-strand or double-strand break canbe introduced in the target site by the polynucleotide guidedpolypeptide, the first and second regions of homology flanking the donorpolynucleotide (e.g. donor DNA) can undergo homologous recombinationwith their corresponding genomic regions of homology resulting inexchange of DNA between the donor and the genome. As such, the providedmethods can result in the integration of the donor polynucleotide (e.g.donor DNA) into the single-strand or double-strand break(s) in thetarget site in the organellar genome, thereby altering the originaltarget site and producing an altered genomic target site.

The donor polynucleotide (e.g. donor DNA) may be introduced by anysuitable means. For example, a plant having a target site can beprovided. The donor polynucleotide (e.g. donor DNA) may be provided byany suitable transformation method including, for example,Agrobacterium-mediated transformation or biolistic particle bombardment.The donor polynucleotide (e.g. donor DNA) may be present transiently inthe cell or it could be introduced via a viral replicon. In the presenceof the guide polynucleotide (e.g., guide RNA), the polynucleotide guidedpolypeptide (e.g., Cas polypeptide) and the target site, the donorpolynucleotide (e.g. donor DNA) can be inserted into the organellargenome.

Donor polynucleotides can be reflective of the commercial markets. Donorpolynucleotides can be reflective of traits for the development of thecrop. Crops and markets of interest can change, and as developingnations open up world markets, new crops and technologies can emergealso. In addition, as the understanding of agronomic traits andcharacteristics such as yield and heterosis increase, the choice ofgenes for transformation can change accordingly.

Methods for Modulating Gene Expression

In some aspects are provided methods for modulating expression (e.g.,transcription) of a target nucleic acid (e.g., a gene) in a host cell ororganelle. The methods can involve contacting the target nucleic acidwith an enzymatically inactive Cas protein (e.g., dead Cas) and a guidepolynucleic acid.

In some aspects, the present disclosure provides a method of selectivelymodulating transcription of a target nucleic acid in a host cell. Themethod can involve introducing into the host cell an enzymaticallyinactive Cas protein (e.g., dead Cas) and a guide polynucleic acid. Theguide nucleic acid and the dead Cas protein can form a complex in thehost cell. The complex can selectively modulate transcription of atarget polynucleic acid (e.g., target DNA) in the host cell ororganelle.

In some aspects, the present disclosure provides for selectivetranscription modulation (e.g., reduction or increase) of a targetnucleic acid in a host cell. Selective modulation of transcription of atarget nucleic acid can reduce or increase transcription of the targetnucleic acid, but may not substantially modulate transcription of anon-target nucleic acid or off-target nucleic acid, e.g., transcriptionof a non-target nucleic acid may be modulated by less than 1%, less than5%, less than 10%, less than 20%, less than 30%, less than 40%, or lessthan 50% compared to the level of transcription of the non-targetnucleic acid in the absence of the guide nucleic acid/enzymaticallyinactive or enzymatically reduced Cas protein complex. For example,selective modulation (e.g., reduction or increase) of transcription of atarget nucleic acid can reduce or increase transcription of the targetnucleic acid by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or greater than90%, compared to the level of transcription of the target nucleic acidin the absence of a guide nucleic acid/enzymatically inactive orenzymatically reduced Cas protein complex.

In some aspects, the disclosure provides methods for increasingtranscription of a target nucleic acid. The transcription of a targetnucleic acid can increase by at least about 1.1 fold, at least about 1.2fold, at least about 1.3 fold, at least about 1.4 fold, at least about1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at leastabout 1.8 fold, at least about 1.9 fold, at least about 2 fold, at leastabout 2.5 fold, at least about 3 fold, at least about 3.5 fold, at leastabout 4 fold, at least about 4.5 fold, at least about 5 fold, at leastabout 6 fold, at least about 7 fold, at least about 8 fold, at leastabout 9 fold, at least about 10 fold, at least about 12 fold, at leastabout 15 fold, at least about 20-fold, at least about 50-fold, at leastabout 70-fold, or at least about 100-fold compared to the level oftranscription of the target polynucleic acid (e.g., target DNA) in theabsence of a guide nucleic acid/enzymatically inactive or enzymaticallyreduced Cas protein complex. Selective increase of transcription of atarget nucleic acid increases transcription of the target nucleic acid,but may not substantially increase transcription of a non-targetpolynucleic acid, e.g., transcription of a non-target nucleic acid canbe increased, if at all, by less than about 5-fold, less than about4-fold, less than about 3-fold, less than about 2-fold, less than about1.8-fold, less than about 1.6-fold, less than about 1.4-fold, less thanabout 1.2-fold, or less than about 1.1-fold compared to the level oftranscription of the non-targeted DNA in the absence of the guidenucleic acid/enzymatically inactive or enzymatically reduced Cas proteincomplex.

In some aspects, the disclosure provides methods for decreasingtranscription of a target nucleic acid. The transcription of a targetnucleic acid can decrease by at least about 1.1 fold, at least about 1.2fold, at least about 1.3 fold, at least about 1.4 fold, at least about1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at leastabout 1.8 fold, at least about 1.9 fold, at least about 2 fold, at leastabout 2.5 fold, at least about 3 fold, at least about 3.5 fold, at leastabout 4 fold, at least about 4.5 fold, at least about 5 fold, at leastabout 6 fold, at least about 7 fold, at least about 8 fold, at leastabout 9 fold, at least about 10 fold, at least about 12 fold, at leastabout 15 fold, at least about 20-fold, at least about 50-fold, at leastabout 70-fold, or at least about 100-fold compared to the level oftranscription of the target polynucleic acid (e.g., target DNA) in theabsence of a guide nucleic acid/enzymatically inactive or enzymaticallyreduced Cas protein complex. Selective decrease of transcription of atarget nucleic acid decreases transcription of the target nucleic acid,but may not substantially decrease transcription of a non-target DNA,e.g., transcription of a non-target nucleic acid can be decreased, if atall, by less than about 5-fold, less than about 4-fold, less than about3-fold, less than about 2-fold, less than about 1.8-fold, less thanabout 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, orless than about 1.1-fold compared to the level of transcription of thenon-targeted DNA in the absence of the guide nucleic acid/enzymaticallyinactive or enzymatically reduced Cas protein complex.

Transcription modulation can be achieved by fusing the enzymaticallyinactive Cas protein to a heterologous sequence. The heterologoussequence can be a suitable fusion partner, e.g., a polypeptide thatprovides an activity that indirectly increases, decreases, or otherwisemodulates transcription by acting directly on the target nucleic acid oron a polypeptide (e.g., a histone or other DNA-binding protein)associated with the target nucleic acid. Non-limiting examples ofsuitable fusion partners include a polypeptide that provides formethyltransferase activity, demethylase activity, acetyltransferaseactivity, deacetylase activity, kinase activity, phosphatase activity,ubiquitin ligase activity, deubiquitinating activity, adenylationactivity, deadenylation activity, SUMOylating activity, deSUMOylatingactivity, ribosylation activity, deribosylation activity, myristoylationactivity, or demyristoylation activity.

A suitable fusion partner can include a polypeptide that directlyprovides for increased transcription of the target nucleic acid. Forexample, a transcription activator or a fragment thereof, a protein orfragment thereof that recruits a transcription activator, or a smallmolecule/drug-responsive transcription regulator. A suitable fusionpartner can include a polypeptide that directly provides for decreasedtranscription of the target nucleic acid. For example, a transcriptionrepressor or a fragment thereof, a protein or fragment thereof thatrecruits a transcription repressor, or a small molecule/drug-responsivetranscription regulator.

The heterologous sequence or fusion partner can be fused to theC-terminus, N-terminus, or an internal portion (i.e., a portion otherthan the N- or C-terminus) of the dead Cas protein.

Methods for Delivery

Any suitable delivery method can be used for introducing thecompositions and molecules of the disclosure into a host cell ororganelle. The compositions (e.g., Cas protein, polynucleotide-guidedpolypeptide, guide polynucleic acid, donor polynucleotide) can bedelivered simultaneously or temporally separated. The choice of methodof genetic modification can be dependent on the type of cell beingtransformed and/or the circumstances under which the transformation istaking place (e.g., in vitro, ex vivo, or in vivo).

A method of delivery can involve contacting a target polynucleotide orintroducing into a cell (or a population of cells) one or more nucleicacids comprising nucleotide sequences encoding the compositions of thedisclosure. Suitable nucleic acids comprising nucleotide sequencesencoding the compositions of the disclosure can include expressionvectors, where an expression vector comprising a nucleotide sequenceencoding one or more compositions of the disclosure can be a recombinantexpression vector.

Non-limiting examples of delivery methods or transformation include, forexample, viral or bacteriophage infection, transfection, conjugation,protoplast fusion, lipofection, electroporation, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

In some aspects, the present disclosure provides methods comprisingdelivering one or more polynucleotides, or one or more vectors asdescribed herein, or one or more transcripts thereof, and/or one orproteins transcribed therefrom, to a host cell or organelle. In someaspects, the disclosure further provides cells produced by such methods,and organisms (such as animals, plants, or fungi) and organellescomprising or produced from such cells. In some embodiments, a Casprotein in combination with, and optionally complexed with, a guidesequence can be delivered to a cell or organelle.

Viral and non-viral based gene transfer methods can be used to introducenucleic acids. Such methods can be used to administer nucleic acidsencoding compositions of the disclosure to cells in culture, or in ahost organism. Non-viral vector delivery systems can include DNAplasmids, RNA (e.g. a transcript of a vector described herein), nakednucleic acid, and nucleic acid complexed with a delivery vehicle, suchas a liposome. Viral vector delivery systems can include DNA and RNAviruses, which can have either episomal or integrated genomes afterdelivery to the cell.

Methods of non-viral delivery of nucleic acids can include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides can be used. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration). The preparation of lipid:nucleic acid complexes,including targeted liposomes such as immunolipid complexes, can be used.

RNA or DNA viral based systems can be used to target specific cells andtrafficking the viral payload to an organelle of the cell. Viral vectorscan be administered directly (in vivo) or they can be used to treatcells in vitro, and the modified cells can optionally be administered(ex vivo). Viral based systems can include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Integration in the host genome can occur with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, which canresult in long term expression of the inserted transgene. Hightransduction efficiencies can be observed in many different cell typesand target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that can transduce orinfect non-dividing cells and produce high viral titers. Selection of aretroviral gene transfer system can depend on the target tissue.Retroviral vectors can comprise cis-acting long terminal repeats withpackaging capacity for up to 6-10 kb of foreign sequence. The minimumcis-acting LTRs can be sufficient for replication and packaging of thevectors, which can be used to integrate the therapeutic gene into thetarget cell to provide permanent transgene expression. Retroviralvectors can include those based upon murine leukemia virus (MuLV),gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV),human immuno deficiency virus (HIV), and combinations thereof.

An adenoviral-based systems can be used. Adenoviral-based systems canlead to transient expression of the transgene. Adenoviral based vectorscan have high transduction efficiency in cells and may not require celldivision. High titer and levels of expression can be obtained withadenoviral based vectors. Adeno-associated virus (“AAV”) vectors can beused to transduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and for in vivo and ex vivogene therapy procedures.

Packaging cells can be used to form virus particles that can infect ahost cell. Such cells can include 293 cells, (e.g., for packagingadenovirus), and .psi.2 cells or PA317 cells (e.g., for packagingretrovirus). Viral vectors can be generated by producing a cell linethat packages a nucleic acid vector into a viral particle. The vectorscan contain the minimal viral sequences required for packaging andsubsequent integration into a host. The vectors can contain other viralsequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions can besupplied in trans by the packaging cell line. For example, AAV vectorscan comprise ITR sequences from the AAV genome which are required forpackaging and integration into the host genome. Viral DNA can bepackaged in a cell line, which can contain a helper plasmid encoding theother AAV genes, namely rep and cap, while lacking ITR sequences. Thecell line can also be infected with adenovirus as a helper. The helpervirus can promote replication of the AAV vector and expression of AAVgenes from the helper plasmid. Contamination with adenovirus can bereduced by, e.g., heat treatment to which adenovirus can be moresensitive than AAV. Additional methods for the delivery of nucleic acidsto cells can be used, for example, as described in US20030087817,incorporated herein by reference.

A host cell can be transiently or non-transiently transfected with oneor more vectors described herein. A cell can be transfected as itnaturally occurs in a subject. A cell can be taken or derived from asubject and transfected. A cell can be derived from cells taken from asubject, such as a cell line. In some embodiments, a cell transfectedwith one or more vectors described herein can be used to establish a newcell line comprising one or more vector-derived sequences. In someembodiments, a cell transiently transfected with the compositions of thedisclosure (such as by transient transfection of one or more vectors, ortransfection with RNA), and modified through the activity of a CRISPRcomplex, can be used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence.

Any suitable vector compatible with the host cell can be used with themethods of the disclosure. Non-limiting examples of vectors includepXT1, pSG5 (Stratagene™) pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia™).

In some embodiments, a nucleotide sequence encoding a guide nucleic acidand/or Cas protein can be operably linked to a control element, e.g., atranscriptional control element, such as a promoter. In someembodiments, a nucleotide sequence encoding a guide nucleic acid and/ora Cas protein can be operably linked to multiple control elements thatallow expression of the nucleotide sequence encoding a guide nucleicacid and/or a Cas protein or chimera.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(e.g., U6 promoter, H1 promoter, etc.; see above).

In some embodiments, compositions of the disclosure can be provided asRNA. In such cases, the compositions of the disclosure can be producedby direct chemical synthesis or may be transcribed in vitro from a DNA.The compositions of the disclosure can be synthesized in vitro using anRNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6polymerase, etc.). Once synthesized, the RNA can directly contact atarget polynucleic acid (e.g., target DNA) or can be introduced into acell using any suitable technique for introducing nucleic acids intocells (e.g., microinjection, electroporation, transfection, etc).

Nucleotides encoding a guide nucleic acid (introduced either as DNA orRNA) and/or a Cas protein (introduced as DNA or RNA) can be provided tothe cells using a suitable transfection technique. Nucleic acidsencoding the compositions of the disclosure may be provided on vectorsor cassettes (e.g., DNA vectors). Many vectors, e.g. plasmids, cosmids,minicircles, phage, viruses, etc., useful for transferring nucleic acidsinto target cells are available. The vectors comprising the nucleicacid(s) can be maintained episomally, e.g. as plasmids, minicircle DNAs,viruses such cytomegalovirus, adenovirus, etc., or they may beintegrated into the target cell genome, through homologous recombinationor random integration, e.g. retrovirus-derived vectors such as MMLV,HIV-1, and ALV.

A Cas protein can be provided to cells as a polypeptide. Such a proteinmay optionally be fused to a polypeptide domain that increasessolubility of the product. The domain may be linked to the polypeptidethrough a defined protease cleavage site, e.g. a TEV sequence, which canbe cleaved by TEV protease. The linker may also include one or moreflexible sequences, e.g. from 1 to 10 glycine residues. In someembodiments, the cleavage of the fusion protein can be performed in abuffer that maintains solubility of the product, e.g. in the presence offrom 0.5 to 2 M urea, in the presence of polypeptides and/orpolynucleotides that increase solubility, and the like. Domains ofinterest include endosomolytic domains, e.g. influenza HA domain; andother polypeptides that aid in production, e.g. IF2 domain, GST domain,GRPE domain, and the like. The polypeptide may be formulated forimproved stability. For example, the peptides may be PEGylated, wherethe polyethyleneoxy group provides for enhanced lifetime in the bloodstream.

The compositions of the disclosure may be fused to a polypeptidepermeant domain to promote uptake by the cell. A number of permeantdomains can be used in the non-integrating polypeptides of the presentdisclosure, including peptides, peptidomimetics, and non-peptidecarriers. For example, a permeant peptide may be derived from the thirdalpha helix of Drosophila melanogaster transcription factorAntennapaedia, referred to as penetratin, which comprises the amino acidsequence RQIKIWFQNRRMKWKK (SEQ ID NO: 172). As another example, thepermeant peptide can comprise the HIV-1 tat basic region amino acidsequence, which may include, for example, amino acids 49-57 ofnaturally-occurring tat protein. Other permeant domains can includepoly-arginine motifs, for example, the region of amino acids 34-56 ofHIV-1 rev protein, nona-arginine, and octa-arginine. The nona-arginine(R9) sequence can be used. The site at which the fusion can be made maybe selected in order to optimize the biological activity, secretion orbinding characteristics of the polypeptide.

The compositions of the disclosure may be produced in vitro or by hostcells, and it may be further processed by unfolding, e.g. heatdenaturation, DTT reduction, etc. and may be further refolded.

The compositions of the disclosure may be prepared by in vitrosynthesis. Various commercial synthetic apparatuses can be used. Byusing synthesizers, naturally occurring amino acids can be substitutedwith unnatural amino acids. The particular sequence and the manner ofpreparation can be determined by convenience, economics, and purityrequired.

The compositions of the disclosure may also be isolated and purified inaccordance with recombinant synthesis methods. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. The compositions can comprise, for example, atleast 20% by weight of the desired product, at least about 75% byweight, at least about 95% by weight, and for therapeutic purposes, forexample, at least about 99.5% by weight, in relation to contaminantsrelated to the method of preparation of the product and itspurification. The percentages can be based upon total protein.

The compositions of the disclosure, whether introduced as nucleic acidsor polypeptides, can be provided to the cells for about 30 minutes toabout 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours,3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16hours, 18 hours, 20 hours, or any other period from about 30 minutes toabout 24 hours, which can be repeated with a frequency of about everyday to about every 4 days, e.g., every 1.5 days, every 2 days, every 3days, or any other frequency from about every day to about every fourdays. The compositions may be provided to the subject cells one or moretimes, e.g. one time, twice, three times, or more than three times, andthe cells allowed to incubate with the agent(s) for some amount of timefollowing each contacting event e.g. 16-24 hours, after which time themedia can be replaced with fresh media and the cells can be culturedfurther.

In cases in which two or more different targeting complexes are providedto the cell (e.g., two different guide nucleic acids that arecomplementary to different sequences within the same or different targetpolynucleic acid (e.g., target DNA)), the complexes may be providedsimultaneously (e.g. as two polypeptides and/or nucleic acids), ordelivered simultaneously. Alternatively, they may be providedconsecutively, e.g. the targeting complex being provided first, followedby the second targeting complex, etc. or vice versa.

An effective amount of the compositions of the disclosure can beprovided to the target polynucleic acid (e.g., target DNA) or cells. Aneffective amount can be the amount to induce, for example, at leastabout a 2-fold change (increase or decrease) or more in the amount oftarget nucleic acid modulation (e.g., expression) observed between twohomologous sequences relative to a negative control, e.g. a cellcontacted with an empty vector or irrelevant polypeptide. An effectiveamount or dose can induce, for example, about 2-fold change, about3-fold change, about 4-fold change, about a 7-fold, about 8-foldincrease, about 10-fold, about 50-fold, about 100-fold, about 200-fold,about 500-fold, about 700-fold, about 1000-fold, about 5000-fold, orabout 10.000-fold change in target gene modulation (e.g., expression).The amount of target gene modulation may be measured by any suitablemethod.

Contacting the cells with a composition of the disclosure can occur inany culture media and under any culture conditions that promote thesurvival of the cells. For example, cells may be suspended in anyappropriate nutrient medium. The culture may contain growth factors towhich the cells are responsive. Growth factors can be molecules that canpromote survival, growth and/or differentiation of cells (e.g., inculture, in the intact tissue), for example, through specific effects ona transmembrane receptor. Growth factors can include polypeptides andnon-polypeptide factors.

In numerous embodiments, the chosen delivery system can be targeted tospecific cell types. In some cases, tissue- or cell-targeting of thedelivery system can be achieved by binding the delivery system totissue- or cell-specific markers, such as cell surface proteins. Viraland non-viral delivery systems can be customized to target tissue orcell-types of interest.

Genome Editing Using a Polynucleotide Guided Polypeptide System

As described herein, the polynucleotide guided polypeptide system can beused in combination with a co-delivered polynucleotide modificationtemplate to allow for editing of an organellar nucleotide sequence ofinterest. Also, as described herein, for each embodiment that uses anRNA guided polypeptide system, a similar polynucleotide guidedpolypeptide system can be deployed where the guide polynucleotide maynot solely comprise ribonucleic acids but wherein the guidepolynucleotide comprises a combination of RNA-DNA molecules or solelycomprises DNA molecules.

Genome modification methods can rely on the homologous recombinationsystem. Homologous recombination (HR) can provide molecular means forfinding genomic DNA sequences of interest and modifying them accordingto the experimental specifications. Homologous recombination can beenhanced by introducing double-strand breaks (DSBs) at selectedendonuclease target sites. Described herein is the use of apolynucleotide guided polypeptide system which can provide flexiblegenome cleavage specificity and can result in a high frequency ofdouble-strand breaks at an organellar DNA target site. This specificcleavage can enable efficient gene editing of a nucleotide sequence ofinterest. The nucleotide sequence of interest to be edited can belocated within or outside the target site recognized and/or cleaved by apolynucleotide guided polypeptide (e.g., a Cas polypeptide).

The term “polynucleotide modification template” can refer to apolynucleotide that can comprise at least one nucleotide modificationwhen compared to the nucleotide sequence to be edited. A nucleotidemodification can be at least one nucleotide substitution, addition ordeletion. Examples of minor genome modifications created by use of apolynucleotide modification template include creation of a mutant allele(e.g., antibiotic resistant rRNA gene) and removal of a target site fora polynucleotide guided polypeptide. Optionally, the polynucleotidemodification template can be flanked by homologous nucleotide sequences,wherein the flanking homologous nucleotide sequences can providesufficient homology to the desired nucleotide sequence to be edited. Thepolynucleotide modification template can be a donor polynucleotide.

In one embodiment, the disclosure provides a method for editing anucleotide sequence in the organellar genome of a cell. The method cancomprise providing a guide polynucleotide (e.g., guide RNA), apolynucleotide modification template, and at least one polynucleotideguided polypeptide (e.g., Cas polypeptide) to an organelle. Thepolynucleotide guided polypeptide can introduce a single-strand ordouble-strand break at a target sequence in the organellar genome of thecell. The polynucleotide modification template can include at least onenucleotide modification of said nucleotide sequence. Cells include, butare not limited to, human, animal, bacterial, fungal, insect, and plantcells as well as organisms and tissues, e.g., plants and seeds, producedby the methods described herein. Cell can be an isolated and purifiedhuman cell. The nucleotide to be edited can be located within or outsidea target site recognized and cleaved by a polynucleotide guidedpolypeptide. In one embodiment, the at least one nucleotide modificationmay not be a modification at a target site recognized and cleaved by apolynucleotide guided polypeptide. In another embodiment, there can beat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500,600, 700, 900 or 1000 nucleotides between the at least one nucleotide tobe edited and the organellar DNA target site.

In another embodiment, the disclosure provides a method for editing anucleotide sequence in the organellar genome of a cell. The method cancomprise providing a guide polynucleotide (e.g., guide RNA), apolynucleotide modification template and at least one polynucleotideguided polypeptide (e.g., Cas polypeptide) to an organelle, wherein saidguide polynucleotide and said polynucleotide guided polypeptide can forma complex that can enable the polynucleotide guided polypeptide tointroduce a single-strand or double-strand break at an organellar targetsite, wherein said polynucleotide modification template comprises atleast one nucleotide modification of said nucleotide sequence.

In another embodiment, the disclosure provides a method for editing anucleotide sequence in the organellar genome of a plant cell. The methodcan comprise introducing a guide polynucleotide (e.g., guide RNA), apolynucleotide modification template, and at least one organellecodon-optimized polynucleotide guided polypeptide (e.g., Cas9polypeptide) into an organelle, wherein the organelle optimizedpolynucleotide guided polypeptide can introduce a single-strand ordouble-strand break at an organellar target sequence, wherein saidpolynucleotide modification template includes at least one nucleotidemodification of said nucleotide sequence.

The nucleotide sequence to be edited can be a sequence that can beendogenous, artificial, pre-existing, or transgenic to the cell that isbeing edited. For example, the nucleotide sequence in the organellargenome of a cell can be a transgene that is stably incorporated into theorganellar genome of a cell. Editing of such transgene may result in afurther desired phenotype or genotype. The nucleotide sequence in thegenome of a cell can also be a mutated or pre-existing sequence that waseither endogenous or artificial from origin such as an endogenous geneor a mutated gene of interest.

In one embodiment, the region of interest can be flanked by twoindependent guide polynucleotide/polypeptide target sequences. Cuttingcan be done concurrently. The deletion event can be the repair of thetwo chromosomal ends without the region of interest. Alternative resultscan include inversions of the region of interest, mutations at the cutsites and duplication of the region of interest.

Methods for Identifying at Least One Plant Cell Comprising in itsOrganellar Genome a Polynucleotide of Interest Integrated at the TargetSite.

Further provided are methods for identifying at least one plant cellcomprising in its organellar genome a polynucleotide of interestintegrated at the target site. A donor polynucleotide can comprise apolynucleotide of interest. A polynucleotide of interest can beintegrated at a target site in a cell (e.g., genome). A variety ofmethods can be used for identifying those plant cells with insertioninto the genome at or near to the target site without using a screenablemarker phenotype. Such methods can be viewed as directly analyzing atarget sequence to detect any change in the target sequence, includingbut not limited to PCR methods, sequencing methods, nuclease digestion,Southern blots, and any combination thereof.

The method can also comprise recovering a plant from the plant cellcomprising a polynucleotide of interest integrated into its organellargenome. The plant may be sterile or fertile. Any polynucleotide ofinterest can be provided, integrated into the plant organellar genome atthe target site, and expressed in a plant.

Polynucleotides of interest can be reflective of the commercial marketsand interests of those involved in the development of the crop. Cropsand markets of interest change, and as developing nations open up worldmarkets, new crops and technologies can emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield,stress tolerance and heterosis increase, the choice of genes fortransformation can change accordingly.

Polynucleotides/polypeptides of interest include, but are not limitedto, herbicide-tolerance coding sequences, insecticidal coding sequences,nematicidal coding sequences, antimicrobial coding sequences, antifungalcoding sequences, antiviral coding sequences, abiotic and biotic stresstolerance coding sequences, or sequences modifying plant traits such asyield, grain quality, nutrient content, starch quality and quantity,nitrogen fixation and/or utilization, and oil content and/orcomposition. polynucleotides of interest can include, but are notlimited to, genes that improve crop yield, polypeptides that improvedesirability of crops, genes encoding proteins conferring resistance toabiotic stress, such as drought, nitrogen, temperature, salinity, toxicmetals or trace elements, or those conferring resistance to toxins suchas pesticides and herbicides, or to biotic stress, such as attacks byfungi, viruses, bacteria, insects, and nematodes, and development ofdiseases associated with these organisms. Genes of interest can include,for example, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. Polynucleotides of interestcan include genes encoding important traits for agronomics, insectresistance, disease resistance, herbicide resistance, fertility orsterility, grain characteristics, and commercial products. Genes ofinterestcan include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affectingphotosynthesis, photorespiration and ATP metabolism.

Commercial traits can also be obtained by expression of proteins encodedon a polynucleotide. A commercial use of transformed plants can be theproduction of polymers and bioplastics. Polynucleotides of interest caninclude genes such as β-ketothiolase, PHBase (polyhydroxybutyratesynthase), and acetoacetyl-CoA reductase can facilitate expression ofpolyhydroxyalkanoates (PHAs).

Polynucleotides/polypeptides that can influence amino acid biosynthesisinclude, for example, anthranilate synthase (AS; EC 4.1.3.27) which cancatalyze the first reaction branching from the aromatic amino acidpathway to the biosynthesis of tryptophan in plants, fungi, andbacteria. In plants, the chemical processes for the biosynthesis oftryptophan can be compartmentalized in the chloroplast. Additional donorsequences of interest can include Chorismate Pyruvate Lyase (CPL) whichcan refer to a gene encoding an enzyme can which catalyze the conversionof chorismate to pyruvate and pHBA. Once example of CPL gene is from E.coli and bears the GenBank accession number M96268.

Polynucleotide sequences of interest may encode proteins involved inproviding disease or pest resistance. By “disease resistance” or “pestresistance” can be intended that the plants can avoid the harmfulsymptoms that are the outcome of the plant-pathogen interactions. Pestresistance genes may encode resistance to pests that have great yielddrag such as rootworm, cutworm, European Corn Borer, and the like.Disease resistance and insect resistance genes such as lysozymes orcecropins for antibacterial protection, or proteins such as defensins,glucanases or chitinases for antifungal protection, or Bacillusthuringiensis endotoxins, protease inhibitors, collagenases, lectins, orglycosidases for controlling nematodes or insects are all examples ofuseful gene products. Genes encoding disease resistance traits includedetoxification genes, such as against fumonisin; avirulence (avr) anddisease resistance (R) genes; and the like. Insect resistance genes mayencode resistance to pests that have great yield drag such as rootworm,cutworm, European Corn Borer, and the like. Such genes include, forexample, Bacillus thuringiensis toxic protein genes; and the like.

An “herbicide resistance protein” or a protein resulting from expressionof an “herbicide resistance-encoding nucleic acid molecule” can includeproteins that confer upon a cell the ability to tolerate a higherconcentration of an herbicide than cells that do not express theprotein, or to tolerate a certain concentration of an herbicide for alonger period of time than cells that do not express the protein.Herbicide resistance traits may be introduced into plants by genescoding for resistance to herbicides that act to inhibit the action ofacetolactate synthase (ALS), for example, the sulfonylurea-typeherbicides, genes coding for resistance to herbicides that can act toinhibit the action of glutamine synthase, such as phosphinothricin orbasta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene andthe GAT gene), HPPD inhibitors (e.g., the HPPD gene) or other suchgenes. The bar gene can encodes resistance to the herbicide basta, theaadA can encode resistance to spectinomycin and streptomycin, the nptIIgene can encode resistance to the antibiotics kanamycin and geneticin,and certain ALS-gene mutants can encode resistance to the herbicidechlorsulfuron.

Sterility genes can also be encoded in an expression cassette orintegrated into the genome. Sterility genes can provide an alternativeto physical detasseling. Examples of genes used in such ways includemale fertility genes such as MS26, MS45, or MSCA1. Maize plants (Zeamays L.) can be bred by both self-pollination and cross-pollinationtechniques. Maize can have male flowers, located on the tassel, andfemale flowers, located on the ear, on the same plant. It canself-pollinate (“selfing”) or cross pollinate. Natural pollination canoccur in maize when wind blows pollen from the tassels to the silks thatprotrude from the tops of the incipient ears. Pollination may be readilycontrolled by suitable methods. The development of maize hybrids canrequire the development of homozygous inbred lines, the crossing ofthese lines, and the evaluation of the crosses. Pedigree breeding andrecurrent selections are two of the breeding methods that can be used todevelop inbred lines from populations. Breeding programs can combinedesirable traits from two or more inbred lines or various broad-basedsources into breeding pools from which new inbred lines are developed byselfing and selection of desired phenotypes. A hybrid maize variety canbe a cross of two such inbred lines, each of which may have one or moredesirable characteristics lacked by the other or which complement theother. The new inbreds can be crossed with other inbred lines and thehybrids from these crosses can be evaluated to determine which havecommercial potential. The hybrid progeny of the first generation can bedesignated F1. The F1 hybrid can be more vigorous than its inbredparents. This hybrid vigor, or heterosis, can be manifested in manyways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the maletassel can be removed from the growing female inbred parent, which canbe planted in various alternating row patterns with the male inbredparent. Consequently, providing that there is sufficient isolation fromsources of foreign maize pollen, the ears of the female inbred can befertilized only with pollen from the male inbred. The resulting seed cantherefore be hybrid (F1) and can form hybrid plants.

Field variation impacting plant development can result in plantstasseling after manual detasseling of the female parent is completed.Or, a female inbred plant tassel may not be completely removed duringthe detasseling process. In any event, the result can be that the femaleplant can successfully shed pollen and some female plants can beself-pollinated. This can result in seed of the female inbred beingharvested along with the hybrid seed which can be normally produced.Female inbred seed may not exhibit heterosis and therefore may not be asproductive as F1 seed. In addition, the presence of female inbred seedcan represent a germplasm security risk for the company producing thehybrid.

Alternatively, the female inbred can be mechanically detasseled bymachine. Mechanical detasseling can be approximately as reliable as handdetasseling, but may be faster and less costly. However, mostdetasseling machines can produce more damage to the plants than handdetasseling. Thus, no form of detasseling may be presently entirelysatisfactory, and a need continues to exist for alternatives whichfurther reduce production costs and to eliminate self-pollination of thefemale parent in the production of hybrid seed.

One method to convey male sterility without mechanical detasseling canbe the use of cytoplasmic male sterility (CMS) genes. Chimericmitochondrial ORFs can be found to lead to male sterility, producingunisex-female plants. The methods described herein could be used tointroduce custom-designed, CMS ORFs into mitochondria of maize eliteinbred lines. Additionally, these methods can provide a means tointroduce the CMS system into other crops; e.g., rice, wheat andsoybean.

The donor polynucleotide may also encode an RNA or double-stranded RNAthat can be complementary to a target gene from a plant pest or plantpathogen. A method of alleviating pest infestation of plants cancomprise, for example, a) identifying a DNA sequence from said pestwhich can be critical either for its survival, growth, proliferation orreproduction, b) cloning said sequence or a fragment thereof in asuitable vector relative to one or more promoters that can transcribesaid sequence to RNA or dsRNA upon binding of an appropriatetranscription factor to said promoters, and/or c) introducing saidvector into the plant. The plant pest can be a nematode. Another methodfor alleviating pest infestation can include, for example, providing: a)DNA sequences which when transcribed yield a double-stranded RNAmolecule that can reduce the expression of an essential gene of a plantsap-sucking insect; b) methods of using such DNA sequences and plants orplant cells transformed with such DNA sequences; and c) the use ofcationic oligopeptides that facilitate the entry of dsRNA or siRNAmolecules in insect cells, such as plant sap-sucking insect cells.

The donor polynucleotide may comprise and/or lead to expression ofantisense sequences complementary to at least a portion of the messengerRNA (mRNA) for a targeted gene sequence of interest; e.g., a target genefrom a plant pest or plant pathogen. Antisense nucleotides can beconstructed to hybridize with the corresponding mRNA. Antisensenucleotides can be targeted to bind a splicing site on a pre-mRNA andmodify the exon content of an mRNA, thereby modulating (e.g.,disrupting) expression of a target gene.

Modifications of the antisense sequences may be made as long as thesequences hybridize to and interfere with expression of thecorresponding mRNA. In this manner, antisense constructions having 70%,80%, or 85% sequence identity to the corresponding antisense sequencesmay be used. Furthermore, portions of the antisense nucleotides may beused to disrupt the expression of the target gene. Generally, sequencesof at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greatermay be used.

The donor polynucleotide can also be a phenotypic marker. A phenotypicmarker can be screenable or a selectable marker that includes visualmarkers and selectable markers whether it is a positive or negativeselectable marker. Any phenotypic marker can be used. Specifically, aselectable or screenable marker can comprise a DNA segment that canallow one to identify, or select for or against a molecule or a cellthat contains it, e.g., under particular conditions. These markers canencode an activity, such as, but not limited to, production of RNA,peptide, or protein, or can provide a binding site for RNA, peptides,proteins, inorganic and organic compounds or compositions and the like.

Examples of selectable markers include, but are not limited to, DNAsegments that comprise restriction enzyme sites; DNA segments thatencode products which provide resistance against otherwise toxiccompounds including antibiotics, such as, spectinomycin, ampicillin,kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) andhygromycin phosphotransferase (HPT); DNA segments that encode productswhich are otherwise lacking in the recipient cell (e.g., tRNA genes,auxotrophic markers); DNA segments that encode products which can bereadily identified (e.g., phenotypic markers such as β-galactosidase,GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan(CFP), yellow (YFP), red (RFP), and cell surface proteins); thegeneration of new primer sites for PCR (e.g., the juxtaposition of twoDNA sequence not previously juxtaposed), the inclusion of DNA sequencesnot acted upon or acted upon by a restriction endonuclease or other DNAmodifying enzyme, chemical, etc.; and, the inclusion of a DNA sequencesrequired for a specific modification (e.g., methylation) that allows itsidentification.

Additional selectable markers include genes that can confer resistanceto herbicidal compounds, such as glyphosate, sulfonylureas, glufosinateammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate(2,4-D).

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important use of transformed plants canbe the production of polymers and bioplastics. Genes such asβ-Ketothiolase, PHBase (polyhydroxyburyrate synthase), andacetoacetyl-CoA reductase can facilitate expression ofpolyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This can be achieved by the expression of such proteinshaving enhanced amino acid content.

The transgenes, recombinant DNA molecules, DNA sequences of interest,and donor polynucleotides can comprise one or more DNA sequences forgene silencing of a target gene; e.g., a target gene in a plant pest orplant pathogen. Methods for gene silencing involving the expression ofDNA sequences in plant can include, but are not limited to,cosuppression, antisense suppression, double-stranded RNA (dsRNA)interference, hairpin RNA (hpRNA) interference, intron-containinghairpin RNA (ihpRNA) interference, transcriptional gene silencing, andmicroRNA (miRNA) interference.

In one embodiment, the targeted mutation can involve use of adouble-strand-break-inducing agent that can induce a double-strand breakin the DNA of the target sequence.

In one embodiment, the targeted mutation can be the result of a guidepolynucleotide/polypeptide induced gene editing as described herein. Theguide polynucleotide/polypeptide induced targeted mutation can occur ina nucleotide sequence that can be located within or outside a genomictarget site that can be recognized and cleaved by a polynucleotideguided polypeptide.

In certain embodiments, a fertile plant can be a plant that can produceviable male and female gametes and can be self-fertile. Such aself-fertile plant can produce a progeny plant without the contributionfrom any other plant of a gamete and the genetic material containedtherein. Other embodiments may involve the use of a plant that may notbe self-fertile, for example, because the plant may not produce malegametes, or female gametes, or both, that are viable or otherwisecapable of fertilization. As used herein, a “male sterile plant” can bea plant that does not produce male gametes that are viable or otherwisecapable of fertilization. As used herein, a “female sterile plant” canbe a plant that does not produce female gametes that are viable orotherwise capable of fertilization. Male-sterile and female-sterileplants can be female-fertile and male-fertile, respectively. A malefertile (but female sterile) plant can produce viable progeny whencrossed with a female fertile plant and that a female fertile (but malesterile) plant can produce viable progeny when crossed with a malefertile plant.

Breeding Methods and Methods for Selecting Plants Utilizing a TwoComponent RNA Guide and Cas Polypeptide System

The present disclosure can find use in the breeding of plants comprisingone or more transgenic traits. Transgenic traits can be randomlyinserted throughout the plant genome as a consequence of transformationsystems based on Agrobacterium, biolistics, or other suitableprocedures. Directed transgene insertion can be used. Site-specificintegration (SSI) can enable the targeting of a transgene to the samechromosomal location as a previously inserted transgene. Custom-designedmeganucleases and custom-designed zinc finger meganucleases can be usedto design nucleases to target specific chromosomal locations, and thesereagents can allow the targeting of transgenes at the chromosomal sitecleaved by these nucleases.

Genetic engineering of eukaryotic genomes, e.g. plant genomes, usinghoming endonucleases, meganucleases, zinc finger nucleases, andtranscription activator-like effector nucleases (TALENs) can require denovo protein engineering for every new target locus. The highlyspecific, polynucleotide guided polypeptide system (e.g., guide RNA/Caspolypeptide system) described herein, can be more easily customizableand can be more useful when modification of many different targetsequences is the goal. The polynucleotide guided polypeptide system canbe a two component system, for example, with its constant proteincomponent, the polynucleotide guided polypeptide (e.g., Caspolypeptide), and its variable and easily reprogrammable targetingcomponent, the guide polynucleotide (e.g., guide RNA or crRNA).

The polynucleotide guided polypeptide system described herein can beespecially useful for genome engineering in circumstances whereendonuclease off-target cutting can be toxic to the targeted cells. Inone embodiment of the polynucleotide guided polypeptide system describedherein, the constant component, a polynucleotide encoding an organelletargeted polynucleotide guided polypeptide, can be stably integratedinto the nuclear genome of the cell. The polynucleotide can encode amodified polynucleotide guided polypeptide comprising an enzymaticallyactive polynucleotide guided polypeptide (e.g., Cas polypeptide) fusedto an organellar transport sequence (e.g., a mitochondrial targetingpeptide or a chloroplast targeting peptide). Expression of thepolynucleotide encoding the modified polynucleotide guided polypeptidecan be under control of a promoter. The promoter can be a constitutivepromoter, a tissue-specific promoter or an inducible promoter, e.g. atemperature-inducible, stress-inducible, developmental stage inducible,or chemically inducible promoter. In the absence of the variablecomponent (e.g., the guide RNA or crRNA), the polynucleotide guidedpolypeptide may not cut the target nucleic acid. In the absence of thevariable component (e.g., the guide RNA or crRNA) the presence of thepolynucleotide guided polypeptide in the plant cell may have little orno consequence. A polynucleotide guided polypeptide system can be usedto create and/or maintain a cell line or transgenic organism capable ofefficient expression of the polynucleotide guided polypeptide.Expression of the polynucleotide guided polypeptide in the cell line ortransgenic organism may have little or no consequence to cell viability.In order to induce cutting at desired genomic sites to achieve targetedgenetic modifications, guide polynucleotides (e.g., guide RNAs orcrRNAs) can be introduced by a variety of methods into cells containingthe stably-integrated and expressed expression cassette for thepolynucleotide guided polypeptide. For example, guide polynucleotide(e.g., guide RNAs or crRNAs) can be chemically or enzymaticallysynthesized, and introduced into the polynucleotide guided polypeptideexpressing cells via direct delivery methods such a particle bombardmentor electroporation. A guide polynucleic acid may be fused to an RNAmolecule that allows for transport into an organelle. Alternatively, aguide polynucleic acid may be fused to an RNA molecule that allows forbinding to a protein that facilitates transport into the organelle.

Alternatively, genes that can efficiently express guide polynucleotides(e.g., guide RNAs or crRNAs) in the target cells can be synthesizedchemically, enzymatically or in a biological system. These genes can beintroduced into the polynucleotide guided polypeptide expressing cells,for example, via direct delivery methods such a particle bombardment,electroporation or biological delivery methods such asAgrobacterium-mediated DNA delivery.

One embodiment of the disclosure can be a method for selecting a plantcomprising an altered target site in its organellar genome. The methodcan comprise a) obtaining a first plant that can comprise at least onepolynucleotide guided polypeptide (e.g., Cas polypeptide) that can betransported into an organelle and can introduce a single-strand ordouble strand break at a target site in the organellar genome. In somecases, the polynucleotide guided polypeptide (e.g., dead Cas) may notcleave a target site. The method can further comprise b) obtaining asecond plant comprising a guide polynucleotide (e.g., guide RNA) thatcan be transported into an organelle and can form a complex with thepolynucleotide guided polypeptide of (a). The method can furthercomprise c) crossing the first plant of (a) with the second plant of(b). The method can further comprise d) evaluating the progeny of (c)for an alteration in the target site. The method can further comprise e)selecting a progeny plant that possesses the desired alteration of saidtarget site. When an enzymatically inactive polynucleotide guidedpolypeptide is used, the method can comprise evaluating and selecting aprogeny with altered target gene regulation or expression.

Another embodiment of the disclosure can be a method for selecting aplant comprising an altered target site in its organellar genome. Themethod can comprise: a) obtaining a first plant comprising at least onepolynucleotide guided polypeptide (e.g., Cas polypeptide) that can betransported into an organelle and can introduce a single-strand ordouble strand break at a target site in the organellar genome. Themethod can further comprise b) obtaining a second plant comprising aguide polynucleotide (e.g., guide RNA) and a donor polynucleotide (e.g.donor DNA). The guide polynucleotide and donor polynucleotide (e.g.donor DNA) can be transported into the organelle. The guidepolynucleotide can form a complex with the polynucleotide guidedpolypeptide of (a). The method can further comprise c) crossing thefirst plant of (a) with the second plant of (b). The method can furthercomprise d) evaluating the progeny of (c) for an alteration in thetarget site. The method can further comprise e) selecting a progenyplant that comprises the donor polynucleotide inserted at said targetsite.

Another embodiment of the disclosure can be a method for selecting aplant comprising an altered target site in its organellar genome. Themethod can comprise selecting at least one progeny plant that comprisesan alteration at a target site in its organellar genome. The progenyplant can be a plant, for example, obtained by crossing a first plantexpressing at least one polynucleotide guided polypeptide (e.g., Caspolypeptide) that can be transported into an organelle to a second plantcomprising a guide polynucleotide (e.g., guide RNA) and optionally adonor polynucleotide (e.g. donor DNA), wherein said guide polynucleotideand said donor polynucleotide (e.g. donor DNA) can be transported intoan organelle, wherein said polynucleotide guided polypeptide canintroduce a single-strand or double strand break at said target site.

A suitable method can be used to identify those cells having an alteredgenome at or near a target site without using a screenable markerphenotype. Such methods can be viewed as directly analyzing a targetsequence to detect any change in the target sequence, including but notlimited to PCR methods, sequencing methods, nuclease digestion, Southernblots, and any combination thereof.

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. For example,amino acid sequence variants of the protein(s) can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations can be used.

Guidance regarding amino acid substitutions not likely to affectbiological activity of the protein can be determined.

Conservative substitutions, such as exchanging one amino acid withanother having similar properties, can be carried out. Conservativedeletions, insertions, and amino acid substitutions may not produceradical changes in the characteristics of the protein. The effect of anysubstitution, deletion, insertion, or combination thereof can beevaluated by screening assays. Assays for double-strand-break-inducingactivity can measure, for example, the overall activity and specificityof the agent on DNA substrates containing target sites.

Sufficient homology or sequence identity can indicate that twopolynucleotide sequences have sufficient structural similarity to act assubstrates for a homologous recombination reaction. The structuralsimilarity can include overall length of each polynucleotide fragment,and the sequence similarity of the polynucleotides. Sequence similaritycan be described by the percent sequence identity over the whole lengthof the sequences, and/or by conserved regions comprising localizedsimilarities such as contiguous nucleotides having 100% sequenceidentity, and percent sequence identity over a portion of the length ofthe sequences.

The amount of homology or sequence identity shared by a target and adonor polynucleotide can vary. For example, the length of sequencehomology may be at least one of the following: 20 bp, 50 bp, 100 bp, 150bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000bp, 1250 bp, 1500 bp, 1750 bp, 2000 bp, 2.5 kb, 3 kb, 4 kb, 5 kb, 6 kb,7 kb, 8 kb, 9 kb or 10 kb. The amount of homology can also be describedby percent sequence identity over the full aligned length of the twopolynucleotides which includes percent sequence identity of at least anyof the following: 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sufficient homologycan include any combination of polynucleotide length, global percentsequence identity, and optionally conserved regions of contiguousnucleotides or local percent sequence identity, for example sufficienthomology can be described as a region of 75-150 bp having at least 80%sequence identity to a region of the target locus. Sufficient homologycan also be described by the predicted ability of two polynucleotides tospecifically hybridize under high stringency conditions.

A variety of methods can be used for the introduction of nucleotidesequences and polypeptides into an organism, including, for example,transformation, sexual crossing, and the introduction of thepolypeptide, DNA, or mRNA into the cell. Methods for contacting,providing, and/or introducing a composition into various organisms caninclude but are not limited to, stable transformation methods, transienttransformation methods, virus-mediated methods, and sexual breeding.Stable transformation can indicate that the introduced polynucleotidecan integrate into the genome of the organism and can be inherited byprogeny thereof. Transient transformation can indicate that theintroduced composition can only temporarily be expressed or present inthe organism.

Protocols for introducing polynucleotides and polypeptides into plantsmay vary depending on the type of plant or plant cell targeted fortransformation, such as monocot or dicot. Suitable methods ofintroducing polynucleotides and polypeptides into plant cells andsubsequent insertion into the plant genome include microinjection,meristem transformation, electroporation, Agrobacterium-mediatedtransformation, direct gene transfer, and ballistic particleacceleration.

Alternatively, polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Such methods caninvolve incorporating a polynucleotide within a viral DNA or RNAmolecule. In some examples a polypeptide of interest may be initiallysynthesized as part of a viral polyprotein, which can be later processedby proteolysis in vivo or in vitro to produce the desired recombinantprotein. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, can involve viral DNA or RNAmolecules. Transient transformation methods include, but are not limitedto, the introduction of polypeptides, such as a double-strand breakinducing agent, directly into the organism, the introduction ofpolynucleotides such as DNA and/or RNA polynucleotides, and theintroduction of the RNA transcript, such as an mRNA encoding adouble-strand break inducing agent, into the organism. Such methodsinclude, for example, microinjection or particle bombardment.

DNA transformation of organellar genomes can be performed in, forexample, plastids and mitochondria (e.g., yeast). Selectable markergenes can include, for example, photosynthesis (atpB, tscA, psaA/B,petB, petA, ycf3, rpoA, rbcL), antibiotic resistance (rrnS, rrnL, aadA,nptII, aphA-6), herbicide resistance (psbA, bar, AHAS (ALS), EPSPS,HPPD) and metabolism (BADH, codA, ARG9, ASA2) genes.

DNA transformation of, for example, the yeast nuclear genome can befacilitated by the development of shuttle vectors that can replicate inE. coli and yeast as autonomous plasmids. Vector systems can includelow-copy-number plasmids and integrative DNA through homologousrecombination.

Methods of the invention can provide transformation efficiency into anorganelle (e.g., mitochondria, plastids) of, for example, at leastabout: 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%transformation efficiency.

In one embodiment, an expression construct of the current disclosure maycomprise a promoter operably linked to a nucleotide sequence encoding aCas gene and a promoter operably linked to a guide RNA. The promoter candrive expression of an operably linked nucleotide sequence in a cell.

The cells having the introduced sequence may be grown or regeneratedinto plants. These plants may then be grown, and either pollinated withthe same transformed strain or with a different transformed oruntransformed strain, and the resulting progeny having the desiredcharacteristic and/or comprising the introduced polynucleotide orpolypeptide identified. Two or more generations may be grown to ensurethat the polynucleotide can be stably maintained and inherited, andseeds harvested.

Any plant can be used, including monocot and dicot plants. Examples ofmonocot plants that can be used include, but are not limited to, corn(Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), maize, wheat (Triticumaestivum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum),switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musaspp.), palm, ornamentals, turfgrasses, and other grasses. Examples ofdicot plants that can be used include, but are not limited to, soybean(Glycine max), canola (Brassica napus and B. campestris), alfalfa(Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsisthaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum),and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato(Solanum tuberosum)etc.

The transgenes, recombinant DNA molecules, DNA sequences of interest,and donor polynucleotides can comprise one or more genes of interest.Such genes of interest can encode, for example, a protein that canprovide an agronomic advantage to the plant.

Also, as described herein, for each example or embodiment that cites aguide RNA, a similar guide polynucleotide can be designed wherein theguide polynucleotide does not solely comprise ribonucleic acids butwherein the guide polynucleotide comprises a combination of RNA-DNAmolecules or solely comprises DNA molecules.

In order to edit organellar genomes with polynucleotide guided (e.g.,RNA guided) methodologies, two molecular components, a polynucleotideguided polypeptide (e.g., Cas protein, Cas9) and a guide polynucleotide(e.g., guide RNA), can be introduced into organelles. The introductionof these components may be accomplished by a combination of a suitableapproache. One approach can be to create a modified polynucleotideguided polypeptide by a translational fusion of the polynucleotideguided polypeptide with an organelle targeting peptide that can allowprotein import into an organelle. Another approach can be to create atranscriptional fusion of a guide polynucleic acid with an RNA moleculethat can be imported into an organelle. For the latter, theconfiguration of imported guide polynucleic acid (e.g., guide RNA) canbe designed to enable appropriate function, i.e., the 5′ end of guideRNA can be accessible to bind with the target site on the organellarDNA. The combination of these two components can be sufficient to editorganellar genomes to create small deletions (e.g., SDN1 modifications)and additions of a few nucleotides at the cleavage sites (e.g., SDN2modifications). To achieve organellar genome editing with more extensiveSDN2 and SDN3 modifications, a polynucleotide modification template canbe introduced into the corresponding organelle.

After creating a designed change in organellar DNA, the next step can beto maintain the edited organellar DNA in the pool of unmodifiedorganellar DNA and to shift the balance among organellar DNA to favorthe maintenance of genome edited organellar DNA. This can be achieved byreducing the amplification of unmodified organellar DNA. In oneapproach, guide polynucleic acids can be designed for multiple targetsites in the unmodified organelle genome. The donor polynucleotide (e.g.donor DNA) can be designed such that these target sites have beenaltered to no longer be recognized by the relevant polynucleotide guidedpolypeptide system(s). Expression of the polynucleotide guidedpolypeptides can result in the introduction of single-strand ordouble-strand breaks into the unmodified organellar DNA and can therebyincrease the proportion of modified genomes. In one variation, cells maybe pretreated with relevant polynucleotide guided polypeptide systems tointroduce cleavages in organellar DNA. The pretreatment can reduce thenumber of organelle DNA molecules available for homologousrecombination.

Embodiments can involve a single guide RNA (sgRNA), i.e., where thevariable targeting domain can be fused to a polynucleotide that containsa tracrRNA sequence. Alternatively, embodiments may involve a duplexguide RNA, i.e., where the variable targeting domain and the tracrRNAsequence are present on separate RNA molecules. The terms “duplex guideRNA” and “dual guide RNA” are used interchangeably herein.

In some cases, protein and/or RNA expression levels can be higher whentransformed into an organelle (e.g., plastid, mitochondria) comparedwith that in nucleus. For example, protein expression level can be atleast about: 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%higher with organelle transformation when compared with nucleartransformation. The expression stability of a transcript can be higherwith organelle transformation compared with nuclear transformation.

EMBODIMENTS

In one embodiment, a polynucleotide encoding an RNA sequence maycomprise an organelle targeting RNA operably linked to a guidepolynucleic acid (e.g., single guide RNA), wherein the guide polynucleicacid can direct a polynucleotide guided polypeptide (e.g., Caspolypeptide; Cas9 polypeptide) to cleave a target sequence present in anorganelle genome. The guide polynucleic acid may be single guide RNA ora duplex guide RNA; for a duplex RNA, each component RNA is operablylinked to an organelle targeting RNA. The RNA sequence may furthercomprise a sequence encoding a polynucleotide guided polypeptide (e.g.,Cas polypeptide; Cas9 polypeptide). The RNA sequence may furthercomprise an RNA cleavage site between the guide polynucleic acid and thesequence encoding a polynucleotide guided polypeptide. The RNA cleavagesite may be at least one selected from the group consisting of: a Csy4cleavage site, a C2c2 cleavage site, a ribozyme cleavage site, an RNAseIII cleavage site, and any combination thereof.

In another embodiment, a cell may comprise any of the polynucleotides ofthe disclosure.

In another embodiment, a cell may comprise any of the abovepolynucleotide, wherein the cell further comprising a polynucleotideencoding a modified polynucleotide guided polypeptide, wherein themodified polynucleotide guided polypeptide comprises a polynucleotideguided polypeptide (e.g., Cas polypeptide; Cas9 polypeptide) operablylinked to an organelle targeting peptide.

In another embodiment, a method for introducing a guide polynucleic acidinto an organelle of a cell may comprise: (a) introducing into a cellany of the above polynucleotides, wherein the polynucleotide is operablylinked to at least one regulatory element; and (b) growing the cellunder conditions in which the polynucleotide is expressed. The methodmay further comprise (c) selecting a cell having an organelle thatcomprises a guide polynucleic acid.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell: (i) a first polynucleotideencoding an RNA sequence comprising an organelle targeting RNA operablylinked to a guide polynucleic acid, wherein the guide polynucleic acidcan direct a polynucleotide guided polypeptide (e.g., Cas polypeptide;Cas9 polypeptide) to cleave a target sequence present in an organellegenome, wherein the polynucleotide is operably linked to at least oneregulatory element; and (ii) a second polynucleotide encoding a modifiedpolynucleotide guided polypeptide, wherein the second polynucleotide isoperably linked to at least one regulatory element, and wherein themodified polynucleotide guided polypeptide comprises a polynucleotideguided polypeptide (e.g., Cas polypeptide; Cas9 polypeptide) operablylinked to an organelle targeting peptide; wherein the organelletargeting RNA of (i) and the organelle targeting peptide of (ii) eachtarget the same organelle; and (b) growing the cell under conditions inwhich the first polynucleotide of (i) and the second polynucleotide of(ii) are both expressed. The method may further comprise (c) selecting acell having an organelle that comprises an altered genome.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell: (i) a first polynucleotideencoding an RNA sequence comprising an organelle targeting RNA operablylinked to a guide polynucleic acid, wherein the guide polynucleic acidcan direct a polynucleotide guided polypeptide (e.g., Cas polypeptide;Cas9 polypeptide) to cleave a target sequence present in an organellegenome, wherein the polynucleotide is operably linked to at least oneregulatory element; and (ii) a third polynucleotide, wherein the thirdpolynucleotide is operably linked to at least one regulatory element,wherein the third polynucleotide encodes an RNA molecule comprising anorganelle targeting RNA operably linked to an RNA sequence encoding apolynucleotide guided polypeptide (e.g., Cas polypeptide; Cas9polypeptide); wherein the organelle targeting RNA of (i) and theorganelle targeting RNA of (ii) each target the same organelle; and (b)growing the cell under conditions in which the polynucleotide of (i) andthe third polynucleotide of (ii) are both expressed. The method mayfurther comprise (c) selecting a cell having an organelle that comprisesan altered genome.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into a cell a polynucleotide encoding anRNA sequence comprising an organelle targeting RNA operably linked to aguide polynucleic acid, wherein the guide polynucleic acid can direct apolynucleotide guided polypeptide (e.g., Cas polypeptide; Cas9polypeptide) to cleave a target sequence present in an organelle genome,wherein the RNA sequence further comprises a second RNA sequenceencoding a polynucleotide guided polypeptide (e.g., Cas polypeptide;Cas9 polypeptide), wherein the polynucleotide is operably linked to atleast one regulatory element; and (b) growing the cell under conditionsin which the polynucleotide of (a) is expressed. The method may furthercomprise (c) selecting a cell having an organelle that comprises analtered genome.

In any of the above methods for altering the genome of an organelle, themethod may further comprise introducing a polynucleotide comprising atleast one donor polynucleotide (e.g., donor DNA) into the organelle,wherein the at least one donor polynucleotide is bounded by at least onehomologous sequence with respect to the organelle genome, whereinintegration of all or part of the at least one donor polynucleotide intothe organelle genome results in removal of the target site of the guidepolynucleic acid. The at least one donor polynucleotide may comprise afirst nucleic acid sequence that is heterologous to the organellegenome, wherein the first nucleic acid sequence is bounded by a secondand a third nucleic acid sequence, wherein the second and the thirdnucleic acid sequences correspond to two adjacent regions of homology inthe organelle genome. The first nucleic acid sequence that isheterologous to the organelle genome may encode a selectable marker. Theselectable marker may be aadA and the selection agent may bespectinomycin or streptomycin. The first nucleic acid sequence that isheterologous to the organelle genome may be operably linked to at leastone regulatory element that is active in the organelle. The second orthe third nucleic acid sequence, or both, may comprise at least onealtered sequence, wherein the at least one altered sequence is alteredwith respect to at least one additional target site in the organellegenome, wherein the at least one altered sequence is not cleavable by atleast one additional guide polynucleic acid, wherein the at least oneadditional guide polynucleic acid can direct a polynucleotide guidedpolypeptide (e.g., Cas polypeptide; Cas9 polypeptide) to cleave the atleast one additional target site in the organelle genome. The at leastone additional target site in the organelle genome may be present in atleast one essential coding region. The polynucleotide introduced intothe organelle may further comprise a fourth nucleic acid sequence,wherein the fourth nucleic acid sequence encodes the at least oneadditional guide polynucleic acid operably linked to a promoter that isactive in the organelle.

In another embodiment, a polynucleotide may encode a modified RNA donorsequence, wherein the modified RNA donor sequence may comprise anorganelle targeting RNA operably linked to a donor RNA. The modified RNAdonor sequence may comprise a reverse transcriptase primer site.

In another embodiment, the cell may comprise the polynucleotide encodingthe modified RNA donor sequence, and further comprise a polynucleotideencoding a modified reverse transcriptase, wherein the modified reversetranscriptase comprises a reverse transcriptase operably linked to anorganelle targeting peptide.

In any of the above methods for altering the genome of an organelle, themethod may further comprise introducing a polynucleotide comprising atleast one donor polynucleotide (e.g., donor DNA) into the organelle,wherein the donor polynucleotide is introduced into the organelle by:(a) introducing into a cell a polynucleotide encoding a modified RNAdonor sequence, wherein the modified RNA donor sequence comprises anorganelle targeting RNA operably linked to a donor RNA, wherein themodified RNA donor sequence comprises a reverse transcriptase primersite, and wherein the polynucleotide is operably linked to at least oneregulatory element; (b) introducing into the cell a polynucleotideencoding a modified reverse transcriptase, wherein the modified reversetranscriptase comprises a reverse transcriptase operably linked to anorganelle targeting peptide, wherein the polynucleotide is operablylinked to at least one regulatory element, wherein the organelletargeting RNA of (a) and the organelle targeting peptide of (b) eachtarget the same organelle; and (c) growing the cell under conditionswherein the polynucleotides of (a) and (b) are both expressed. Themethod may further comprise (d) selecting a cell having an organellethat comprises an altered genome.

In another embodiment, a method for altering the genome of an organellemay comprise: (a) introducing into an organelle the following: (i) afirst polynucleotide encoding at least one guide polynucleic acid,wherein the at least one guide polynucleic acid can direct apolynucleotide guided polypeptide (e.g., Cas polypeptide; Cas9polypeptide) to cleave at least one target sequence present in anorganelle genome; (ii) a second polynucleotide encoding a polynucleotideguided polypeptide (e.g., Cas polypeptide; Cas9 polypeptide), whereinthe polynucleotide guided polypeptide, when associated with the guidepolynucleic acid (e.g., guide RNA), can cleave the at least one targetsequence; (iii) optionally, a third polynucleotide encoding at least onehomologous organelle DNA sequence, wherein the at least one homologousorganelle DNA is of sufficient size for homologous recombination,wherein integration of the at least one homologous organelle DNAsequence into the organelle genome results in removal of the at leastone target sequence; (iv) optionally, a fourth polynucleotide encodingat least one selectable marker or at least one screenable marker, orboth, wherein the sequence encoding the at least one selectable marker,or at least one screenable marker, or both, is operably linked to apromoter that is functional in the organelle; and (v) optionally, afifth polynucleotide encoding an origin of replication that isfunctional in the organelle; and (b) growing a cell comprising theorganelle of (a) under conditions in which the first polynucleotide of(i) and the second polynucleotide of (ii) are each expressed. The methodmay further comprise a step (c) of selecting a cell having an organellethat comprises an altered genome. The method may further comprise a step(d) of selecting a cell that is homoplasmic for the altered genome ofthe organelle. The third polynucleotide of (iii) may comprise a sixthand a seventh polynucleotide, wherein the sixth and the seventhpolynucleotides correspond to two adjacent regions of homology in theorganelle genome, wherein the sixth and seventh polynucleotides areseparated by a sequence that is heterologous to the organelle DNA. Thesequence that is heterologous to the organelle DNA may comprise at leastone selected from the group consisting of: the first polynucleotide of(i), the second polynucleotide of (ii), the fourth polynucleotide of(iv), an eighth polynucleotide, and any combination thereof, wherein theeighth polynucleotide encodes an RNA that is heterologous to theorganelle or comprises a non-coding sequence (e.g., a regulatorysequence, such as a promoter) that is heterologous to the organelle, orboth. The RNA that is heterologous to the organelle may be at least oneselected from the group consisting of: an mRNA, a functional RNA, andany combination thereof. The functional RNA may be at least one selectedfrom the group consisting of: guide RNA, siRNA, miRNA, dsRNA, tRNA,rRNA, and any combination thereof. At least one selected from the groupconsisting of: the first polynucleotide of (i), the secondpolynucleotide of (ii), the fourth polynucleotide of (iv), the fifthpolynucleotide of (v), and any combination thereof, may be locatedoutside the region bounded by the sixth and the seventh polynucleotide.The fifth polynucleotide of (v) may encode a plastid origin ofreplication, a mitochondrial origin of replication, or both. The plastidorigin of replication may correspond to DNA sequence from a plastid rRNAintergenic region.

In any of the methods described herein, one or more of thepolynucleotides described herein may be present on a recombinant DNAconstruct.

In any of the methods described herein, the method may comprise morethan one such recombinant DNA construct.

In any of the methods described herein, the recombinant DNA constructmay further comprise a ninth and tenth polynucleotide, wherein the ninthand tenth polynucleotides have 100 percent sequence identity to eachother, and further wherein the ninth and tenth polynucleotides arearranged as direct repeats in the recombinant DNA construct. The ninthand tenth polynucleotides may have at least 20, 21, 22, 23, 24, 25, 30,40, 50, 60, 70, 80, 90 or 100 nucleotides of 100 percent sequenceidentity to each other. The recombinant DNA construct may be linear, andthe ninth and tenth polynucleotides may be present at the 5′ and 3′ endsof the recombinant DNA construct, respectively.

In any of the methods described herein for altering the genome of anorganelle, the recombinant DNA construct may be linear, single-strandedand operably linked to a modified VirD2 protein. The modified VirD2protein may comprise a VirD2 protein operably linked to an organelletargeting peptide, wherein the modified VirD2 protein has also beenmodified such that at least one native nuclear localization sequence ofthe VirD2 protein is no longer functional.

In the above methods for altering the genome of an organelle, therecombinant DNA construct may be operably linked to at least onemodified VirE2 protein. The at least one modified VirE2 protein maycomprise a VirE2 protein operably linked to an organelle targetingpeptide, wherein the at least one modified VirE2 protein has also beenmodified such that at least one native nuclear localization sequence ofthe VirE2 protein is no longer functional.

In any of the methods described herein for altering the genome of anorganelle, the recombinant DNA construct may be operably linked to atleast one modified RecA protein. The at least one modified RecA proteinmay comprise a RecA protein operably linked to an organelle targetingpeptide.

In any of the methods described herein for altering the genome of anorganelle, the recombinant DNA construct may be operably linked to atleast one chimeric polypeptide. The at least one chimeric polypeptidemay comprise an organelle targeting peptide and a cell penetratingpeptide and optionally, a DNA-binding polypeptide.

In another embodiment, a method for altering the genome of an organellemay comprise using of both a site-directed nuclease (e.g., TALENS,Zinc-Finger Nuclease or Meganuclease) and a polynucleotide guidedpolypeptide. The initial cleavage of the organelle genome may be done bya site-directed nuclease (e.g., TALENS, Zinc-Finger Nuclease,Meganuclease), to facilitate homologous recombination with a donorpolynucleotide. The donor polynucleotide may contain modified targetsites that are not recognized by a polynucleotide guided polypeptide. Ahomoplasmic state may be facilitated by cleavage of the unmodifiedorganelle genomes at the target sites by treatment with a polynucleotideguided polypeptide. In another embodiment, any of the above methods mayfurther comprise introducing into the organelle a polynucleotideencoding at least one marker selected from the group consisting of: apositive selectable marker, a negative selectable marker, a screenablemarker, and any combination thereof. The positive selectable marker maybe an herbicide tolerance protein. The herbicide tolerance protein maybe at least one selected from the group consisting of: a4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide, an acetyl coenzyme A carboxylase(ACCase), and any combination thereof. The method may further involvegrowing the cell in the presence of a positive selection agent andselecting a cell that is homoplasmic for the altered genome of theorganelle. Optionally, the method may further involve growing the cellin the absence of the positive selection agent, followed by selecting acell that lacks a non-integrated recombinant DNA construct.Alternatively, the method may further involve growing the cell in theabsence of the positive selection agent, followed by growing the cell inthe presence of a negative selection agent, followed by selecting a cellthat lacks a non-integrated recombinant DNA construct. In the method,the cell may be a plant cell, the organelle may be a plastid, and themethod may further involve regenerating a plant from the plant cellcomprising an altered organelle genome. The plant cell may be monocotcell, e.g., a maize cell. The plant cell may be a dicot cell, e.g., asoybean cell.

In another embodiment, a method for altering a genome of an organellemay comprise: (a) introducing into an organelle of a cell the following:(i) at least one guide RNA, wherein the at least one guide RNA directs apolynucleotide guided polypeptide to cleave at least one target sequencepresent in the genome of the organelle; (ii) a polynucleotide guidedpolypeptide, wherein the polynucleotide guided polypeptide, whenassociated with the at least one guide RNA, cleaves the at least onetarget sequence; and (iii) a replacement DNA; and (b) selecting a cellcomprising an organelle comprising the replacement DNA. The replacementDNA of step (a) part (iii) may comprise fragments of organellar DNA or acomplete organellar DNA from a cultivar, line, sub-species and otherspecies and is distinct from the genome of the organelle of step (a).The replacement DNA may be lacking the at least one target sequence.Additionally, after step (a) part (ii) and prior to step (a) part (iii),a cell may be selected in which the genome of the organelle has beeneliminated.

In another embodiment, the guide polynucleic acid in the methods andcompositions of matter described herein may comprise the following: i)at least 17 nucleotides that are complementary to at least 17nucleotides of a target polynucleic acid, wherein said targetpolynucleic acid is located in the genome of an organelle; and ii) aregion that contacts a polynucleotide-guided polypeptide. The guidepolynucleic acid may comprise one or more RNA bases. The guidepolynucleic acid may be a guide RNA. The guide polynucleic acid may be adual guide RNA. The guide polynucleic acid may be a single guide RNA.

In another embodiment, the polynucleotide-guided polypeptide in themethods and compositions of matter described herein may be selected fromthe group consisting of: a Cas9 protein, a MAD2 protein (U.S. Pat. No.10,011,849; herein incorporated by reference), a MAD7 protein (U.S. Pat.No. 9,982,279; herein incorporated by reference), a CRISPR nuclease, anuclease domain of a Cas protein, a Cpf1 protein, an Argonaute, modifiedversions thereof, and any combination thereof. The sequence encoding thepolynucleotide-guided polypeptide may be codon-optimized for a human, ayeast, an alga, or a plant species.

In any of the methods described herein for altering the genome of anorganelle, the method may further involve growing the cell in thepresence of a positive selection agent and selecting a cell that ishomoplasmic for the altered genome of the organelle. The method mayfurther involve: (i) growing the cell in the absence of the positiveselection agent, followed by selecting a cell that lacks anon-integrated recombinant DNA construct; or (ii) growing the cell inthe absence of the positive selection agent, followed by growing thecell in the presence of a negative selection agent, followed byselecting a cell that lacks a non-integrated recombinant DNA construct.

In any of the methods described herein that involve a guide polynucleicacid and a polynucleotide guided polypeptide, the method may comprise anincrease in transformation efficiency of at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%, as compared tothe corresponding method lacking the guide polynucleic acid, thepolynucleotide guided polypeptide, or lacking both.

In any of the methods described herein that involve a guide polynucleicacid and a polynucleotide guided polypeptide, the method may comprise adecrease in the amount of time required to achieve a homoplasmic state,wherein the decrease is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90%, as compared to the amount of time required for the correspondingmethod lacking the guide polynucleic acid, the polynucleotide guidedpolypeptide, or lacking both.

In another embodiment, a recombinant DNA construct (e.g., for use in anyof the methods described herein) may comprise any one or more of thepolynucleotides described herein.

In another embodiment, a cell may comprise an organelle, wherein theorganelle may comprise at least one of the above recombinant DNAconstructs. The cell may be selected from the group consisting of: ayeast cell, an algal cell, a plant cell, an insect cell, a non-humananimal cell and a mammalian tissue culture cell.

In another embodiment, a plant or seed may comprise any of the aboveorganelles, cells or recombinant DNA constructs.

In another embodiment, a cell comprising an organelle with an alteredgenome may be produced by any of the above methods. The cell may beselected from the group consisting of: a yeast cell, an algal cell, aplant cell, an insect cell, a non-human animal cell and a mammaliantissue culture cell.

In another embodiment, a method may alter the genome of an organelle ina cell, wherein the cell is a plant cell. Furthermore, a plant may beregenerated from the plant cell comprising an organelle with an alteredgenome, wherein the regenerated plant comprises an organelle with analtered genome. Also, a plant (e.g., progeny plant) or seed may beproduced from the regenerated plant, wherein the plant or seed comprisesan organelle with an altered genome.

In any of the above embodiments involving guide polynucleic acid (e.g.,guide RNA), the guide polynucleic acid may be a single guide RNA(unimolecular) or a duplex guide RNA (bimolecular). In any embodimentinvolving multiple guide RNAs, the multiple guide RNAs may be singleguide RNAs, duplex guide RNAs, or both.

In any of the above embodiments, multiple guide RNAs (and/or otherheterologous RNAs) may be encoded on separate transcription units or maybe encoded on a polycistronic transcription unit. A guide RNA may beprocessed from a polycistronic RNA after transcription; e.g., by use ofan RNA cleavage site (e.g., Csy4; C2c2), a ribozyme cleavage site, apolynucleotide guided polypeptide cleavage site or the presence of atRNA sequence. A guide RNA may be processed from a polycistronic RNA byhaving a first tRNA sequence 5′ to the guide RNA and a second tRNAsequence 3′ to the guide RNA. Multiple guide RNAs may be arrayed withmultiple tRNA sequences (at each guide RNA 5′ and 3′ end) for processingfrom a polycistronic RNA.

In any of the above embodiments, the polynucleotide (e.g., donor DNA,donor RNA) that can be introduced into the organelle may comprise atleast one selected from the group consisting of: an expression cassetteencoding a polynucleotide of interest and an expression cassetteencoding a polycistronic transcript that comprises multiplepolynucleotides of interest; e.g., a polycistronic transcript comprisingmultiple protein-coding regions, multiple functional RNAs, or acombination of both. The polynucleotide of interest may be heterologouswith respect to the genome of the organelle.

In any of the above methods for altering the genome of an organelle tocontain a heterologous polynucleotide, the heterologous polynucleotidemay encode at least one selected from the group consisting of: anherbicide tolerance protein, a pesticidal protein, an accessory proteinthat binds to a pesticidal protein, a dsRNA, a siRNA, a miRNA, and anycombination thereof, wherein the dsRNA, the siRNA and the miRNA cansuppress at least one target gene present in a plant pest. The herbicidetolerance protein may be at least one selected from the group consistingof: a 4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide, an acetyl coenzyme A carboxylase(ACCase), and any combination thereof. The pesticidal protein may be atleast one selected from the group consisting of: Cry1Ac, Cyt1Aa, Cry1Ab,Cry2Aa, Cry1l, Cry1C, Cry1D, Cry1E, Cry1Be, Cry1Fa and Vip3A. Theaccessory protein that binds to a pesticidal protein may be at least oneselected from the group consisting of: a 20 kDa accessory protein and a19 kDa accessory protein. The dsRNA, the siRNA and the miRNA cansuppress at least one target gene selected from the group consisting of:proteasome A-type subunit peptide (Pas-4), ACT, SHR, EPIC2B, PnPMAI, andany combination thereof. The heterologous polynucleotide may be operablylinked to at least one regulatory element that is active in anorganelle. The at least one regulatory element may be selected from thegroup consisting of: a maize clpP promoter combined with a maize clpP5′-UTR, a maize clpP promoter combined with a 5′-UTR from gene 10 ofbacteriophage T7, a tomato psbA promoter is combined with a 5′-UTR fromgene 10 of bacteriophage T7, a tomato rm16 promoter combined with amodified accD 5′-UTR, and any combination thereof. The cell may be aplant cell, wherein the organelle is a plastid (e.g., a chloroplast),and wherein the method further comprises regenerating a plant from theplant cell comprising an altered organelle genome. The plant cell may bea soybean cell.

In any of the above methods for altering the genome of an organelle tocontain a heterologous polynucleotide, the heterologous polynucleotidemay be flanked by direct repeat sequences. The direct repeat sequencesmay have at least 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500 or 600 nucleotides of 100 percent sequenceidentity to each other. The direct repeat sequences may comprise asite-specific recombinase site (e.g., loxP, attP, attB). Theheterologous polynucleotide may encode at least one marker selected fromthe group consisting of: a positive selectable marker, a negativeselectable marker, a screenable marker, and any combination thereof.Optionally, the method may further involve growing the cell in theabsence of the positive selection agent, followed by selecting a cellthat is homoplasmic for organelles that lack the heterologouspolynucleotide. Alternatively, the method may further involve growingthe cell in the presence of a negative selection agent, followed byselecting a cell that is homoplasmic for organelles that lack theheterologous polynucleotide. Optionally, the method may involve growingthe cell under conditions in which a heterologous site-specificrecombinase (e.g., Cre, phiC31, Bxb1) is expressed in the organelle.

In the above embodiments, the target organelle may be a plastid (e.g.,chloroplast) or a mitochondrion. The organelle targeting polynucleotidemay be tRNA, viroid RNA or eIF4E RNA.

In the above embodiments, expression of an antibiotic marker gene may beused in conjunction with antibiotic selection for obtaining (andselecting) a plastid or mitochondrial transformation event (e.g., ahomoplasmic event). The polynucleotide comprising the donorpolynucleotide (e.g., donor DNA) may also comprise an expressioncassette for the antibiotic marker gene; the expression cassette may bewithin the donor polynucleotide region (i.e., for integration into theorganelle genome) or outside the donor polynucleotide region.

EXAMPLES

The present disclosure is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating embodiments, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Such modifications are also intended to fall within thescope of the appended claims.

Experiments typically involve a single guide RNA (sgRNA), i.e., wherethe variable targeting domain is fused to a polynucleotide that containsa tracrRNA sequence. Alternatively, experiments may involve a duplexguide RNA, i.e., where the variable targeting domain and the tracrRNAsequence are present on separate RNA molecules.

Example 1 Targeting Cas9 and Guide RNA into Yeast Mitochondria

To create the Cas9 protein for mitochondrial genome editing, a proteinfunctional in nuclear genome editing is modified by fusing amitochondrial targeting peptide at the amino terminal end and bydeleting any NLS (nuclear localization signal) elements. The organelletargeting peptides of the ATPase beta subunit and the 70 KD protein areused for the modification, creating mCas9-A (encoded by SEQ ID NO: 1)and mCas9-B (encoded by SEQ ID NO: 2), respectively. Each polynucleotideencoding a modified Cas9 is cloned into a yeast shuttle vector withexpression of the polynucleotide under control of the Gall promoter,whose activity is induced by galactose as a carbon source in the media.

To create guide RNA for mitochondrial genome editing, the tRNA^(Lys)(tRK1 and modified tRK2 forms) that can be imported into mitochondria isused. Several versions of fusion RNA between tRNA and guide RNA aremade. One approach is to fuse guide RNA to the 5′ end of the tRNA (SEQID NO: 3 and 4). To suppress 5′ end cleavage by RNAse P, the first baseof tRNA is modified in an alternative construct to prevent the pairingwith the corresponding base on the acceptor stem of the tRNA (SEQ ID NO:5 and 6). The second approach is to replace the intron of tRK2 withefficient mitochondrial import in the backbone of tRK2-2 and tRK1 (SEQID NO: 7 and 8, respectively). The third approach is to use the factthat tRK1 (tRNA^(Lys)) can be split into two molecules that togetherretain the property of mitochondrial import. In this case, guide RNA isfused to the 5′ end of second half of the tRK1 in the region calledvariable loop in tRNA structure in a manner that retain the secondarystructure of the tRNA splicing site (SEQ ID NO: 9). The guide RNA fusedwith B form (SEQ ID NO: 10) is co-expressed with A form to facilitateco-import into mitochondria.

A variation of creating synthetic guide RNA with RNA that serves as theefficient vehicle for mitochondrial import is to use the combination ofF-hairpin and D-arm structures of tRK1. These structures are shown tofacilitate import into mitochondria. In this approach, guide RNA isplaced between two structures (SEQ ID NO: 11) or fused with one of themat the 5′ or 3′ ends (e.g. SEQ ID NO: 12 and 13).

For the site-specific cleavage sites, the following mitochondrialsequences were identified as target sites for guide RNA; the guide RNAvariable targeting domain is shown below:

1. (cytochrome b gene) (SEQ ID NO: 14) ACTGATAGAAGTGTAGTAAG_2. (COX1 gene) (SEQ ID NO: 15) ATGATTATTGCAATTCCAAC 3. (COX1 gene)(SEQ ID NO: 16) ATTCCACGATACTTACTACG 4. (COX2 gene) (SEQ ID NO: 17)TCAGCAACACCAAATCAAGA

Each of the above variable targeting domains precedes a PAM sequence.SEQ ID NO: 14-17 precede the following PAM sequences: AGG, AGG, TGG andAGG, respectively.

Eleven nucleotides from the 3′ end of each underlined sequence (adjacentto the PAM sequence), which are considered critical for Cas9 target siterecognition, are unique to the yeast mitochondrial genome based on blastanalyses. Each of the above variable targeting domains is fused at the3′ end with a tracrRNA sequence for Cas9 recognition (SEQ ID NO: 18).Polynucleotides encoding each engineered guide RNA are expressed in thenucleus under control of the SNR52 promoter and the SUP4 terminationelement (SEQ ID NO: 19 and 20, respectively). In this experiment, ayeast shuttle vector for transformation is used. For example, SNR52expression cassettes are cloned into a yeast expression vector such as p416-Gall (URA3+, multicopy plasmid purchased from ATCC). Expressioncassettes encoding mitochondrial targeted Cas9 (“mCas9”) are cloned intothe Sall-Xhol sites of centromeric p 415-galL vector (LEU+) withexpression under control of the GalL promoter whose activity is inducedby galactose in the media as sole carbon source. Vectors are transformedinto a yeast strain allowing auxotropy selection such as BY4733 (mat a)line, and selected for Leu and Ura independent growth.

The transformants of each and/or the combination of mCas9 and guide RNAconstructs are selected on media selective for corresponding auxotropyas single colony lines. The expression of mCas9 endonuclease is inducedby shifting media to the one containing galactose as sole carbon source.Cells derived from single colonies are grown in the inducing media forseveral generations. These lines are analyzed for genome editingefficacies at the molecular level. Cells from multiple lines of eachconstruct and each construct combination are combined together and theirDNA are isolated by using standard DNA isolation protocols such as byusing Yeast DNA Extraction Kit from TheromoFisher (cat#78870). Using PCRprimer sets specific to corresponding genome editing sites, DNA at eachediting site is amplified by PCR reaction. PCR products are subjected tohigh-throughput sequencing such as by using Illumina HiSeq protocolsprovided by the manufacture. The frequency of site-specific mutations ateach target sit is evaluated in comparison with corresponding controlconstructs. The efficacy of genome editing is also analyzed at thefunctional level. After obtaining single colony lines, each line isfurther grown for additional generations in non-selective glucose mediato promote a homoplasmic state of the mitochondrial genome. Yeast cellsare plated on glucose media such as YD medium. Single colonies aretransferred to the glycerol media such as YG medium by replica plating.The efficacy of genome editing is evaluated by the output frequency ofcolonies incapable of growth on glycerol media, i.e. deficient inrespiration due to the mutations in cob, cox1 and cox2 genes,respectively.

The next step of organellar genome editing is to create a dominant andsustainable state of the edited DNA in mitochondria, which initiallycontains a pool of multiple, if not hundreds of, unedited DNA. This isachieved by extending the period of enzymatic reactions of site-specificmodifications in organelles. Depending on several factors such as theimport efficiency of mCas9 and guide RNA into mitochondria, and theaffinity between guide RNA, imported Cas9 and target sites, the lengthof the extended period suitable for each modification of an organellevaries. To assess the effect of extended periods, yeast linestransformed with appropriate mCas9 and guide RNA pairs are grown inselective media for corresponding constructs over a time course ofhours, days and weeks. Then, each culture is subjected to evaluation atthe molecular as well as functional levels as described above. Theperiod of enzymatic states sufficient for the maintenance and phenotypicexpression of edited mitochondrial genomes over generations isdetermined from the time course experiments.

Example 2 Targeting Cas9, Guide RNA and Donor DNA into YeastMitochondria

In order to edit organellar genomes precisely at the nucleotide level,donor DNA (comprising a polynucleotide modification template) is addedto the site-specific endonuclease system. In one approach, donor DNA isintroduced into mitochondria in combination with Cas9 and guide RNA;Cas9 and guide RNA are introduced into mitochondria as described inExample 1. In this example, the donor DNA is designed to create aspecific mutation in the 15S rRNA gene in the mitochondrial genome toconfer paromomycin resistance. The nucleotide substitution (C-to-G) atposition 1514 can confer paromycin resistance. To create the donor DNAwith the resistance allele, one primer pair is designed to carry thecorresponding substitution (SEQ ID NO: 21). PCR amplification isperformed by using the primer set (SEQ ID NO: 21 and 22) and yeast totalDNA as substrate following standard PCR protocols. The resulted templateDNA is transformed into mitochondria via DNA transformation procedures,such as biolistic methods. For transformation with donor DNA, the cellsexpressing Cas9 and guide RNA as described in Example 1 are used withthe exception that the guide RNA is designed to cleave the vicinity ofthe paromomycin-resistance site of mitochondrial DNA as exemplified inSEQ ID NO: 23. The guide RNA is so designed that the cleavage site iscovered by the donor DNA with overlapping sequences sufficient forhomologous recombination at the both ends but the donor DNA is notrecognized as the substrate for site-specific endonuclease activities.For instance, the donor DNA is modified to not include the PAM sequencethat is targeted by the corresponding guide RNA. The variable targetingdomain of the guide RNA is fused at the 3′ end with tracrRNA sequencefor association with Cas endonuclease; guide RNA expression constructsare made by using tRNA^(Lys) derived methods described in Example 1.

After transformation with donor DNA, cells are pooled together and grownin galactose media to induce Cas9 protein for several generations,following the favorable amplification of the engineered DNA by addinggradually increasing amount of paromomycin in the media over additionalgenerations. Cells are plated to make single colonies. Single coloniesare replica plated on media with glycine as the sole carbon source inthe presence and absence of paromomycin to identify paromomycinresistant colonies. The efficiency of genome editing by this method isshown by an increased rate of producing paromomycin resistant cells withtemplate DNA in comparison to control cells not transformed with donorDNA. Gene editing is confirmed by sequencing of the engineered site.

A subsequent genome editing step is performed to eliminate organellarDNA that does not carry the designed modification. This is achieved byany of several approaches. One approach is to expose cells underpositive selection pressure as described above. Another approach is toeliminate or reduce the replication rate of unmodified organelle DNA.This can be achieved by cleaving unmodified DNA by use of site-specificendonucleases such as zinc finger proteins, TALEN and Cas9 systems. Inthe Cas9 approach, expression of specific guide RNAs is used to cleaveunmodified organellar DNA and thereby increase the population ofmodified DNA.

Example 3 Replacement of Endogenous Organellar DNA

This is an alternative method for modification of an organellar genome.In this approach, the first step is to reduce or eliminate theendogenous organellar DNA by using site-specific endonucleases such asCas9 systems. At the same time or subsequently, a replacement organellarDNA is introduced. The replacement DNA can be fragments of organellarDNA or complete organellar DNA that convey a new genotype andcorresponding trait(s) when transformed into the organelle. In the caseof organellar DNA fragments, they can be integrated into the remainingorganellar DNA by homologous recombination. In the case of completeorganellar DNA replacement, the replacement DNA can be isolated fromcultivars, lines, sub species and other species which possess DNAcompositions distinct from the endogenous organellar DNA of recipientcells. One requirement of the replacement DNA can be to contain a DNAelement functioning as a DNA replication origin in the recipientorganelles. The replacement DNA can also be synthesized partially and/orcompletely. When replacement DNA is created in vitro, it can be a linearDNA with the inverted repeat sequence at the ends. The ends canfacilitate homologous recombination in vitro or in vivo to createcircular DNA for replication of organellar DNA in cells. The DNA createdin vitro can also include exogenous DNA elements such as ones to allowselected amplification in bacterial cells.

To reduce or eliminate mitochondial DNA, yeast cells are exposed toprolonged expression of guide RNA and Cas9 protein that are designed tobe imported into mitochondria as described in Example 1 or to besynthesized directly in organelles as described in Example 4. The targetsites are chosen to be unique to the endogenous mitochondrial DNA andnot present in nuclear genome to reduce the chance of any damageoccurring on nuclear genomes when taking the method described inExample 1. The target sites are also chosen to not be present in thereplacement DNA.

Multiple cleavage sites enhance the rate of displacing endogenousorganellar DNA. This can be attained by expressing multiple guide RNAstargeting different unique sequences in the endogenous mitochondrial DNA(e.g., see target sites of Example 1). After Cas9/guide RNA treatment,yeast cells that have lost mitochondrial DNA are identified by lack ofrespiration, inability to grow on media with glycerin as sole carbonsource and the lack of mitochondrial DNA. The resulting rho⁰ conditioncan also be confirmed by absence of the mitochondrial DNA band in a CsClgradient through the method described in Example 1. Once mitochondrialDNA is deleted, cells are then transformed with replacement DNA createdin vitro or in vivo; e.g., mitochondrial DNA derived from differentlines or species with traits distinct from the recipient cells. In thisexample, mitochondrial DNA from antibiotic resistant lines (e.g.IL8-8C/R53) is isolated and transformed into recipient cells that lackthe resistant trait by using the transformation methods described inExample 2. Mitochondrial DNA for use in transformation can also becreated by PCR amplification of organellar DNA by use of a primer setwhose 3′ ends are complementary with each other, sufficient forannealing in vivo. The resulted linear DNA molecules are transformedinto mitochondria. Homologous recombination activity present in theorganelle creates circular organellar DNA upon transformation.Alternatively, DNA for transformation can be created synthetically in alinear as well as a circular form.

Example 4 Introduction into Yeast Mitochondria of Donor DNA andExpression Cassettes for Cas9 and Guide RNA

In this example, a DNA plasmid (“Edit Plasmid”) that can replicate in anorganelle and encodes components of a site-specific endonuclease systemsuch as Cas9, guide RNA and donor DNA is directly introduced into anorganelle. The delivery of nucleic acids and proteins can beaccomplished by utilizing methods such as bombardment (“biolistics”),electroporation and other suitable methods.

In yeast, DNA in a circular form with bacterial vector sequence (pBR322)can be transformed into mitochondria by utilizing a biolistic method.The resulted cells were crossed with a line carrying a point mutation inmitochondrial DNA. They showed that the point mutation was recovered byrecombination between the plasmid DNA and mitochondrial DNA. Forefficient genome editing, a plasmid DNA to be transformed into yeastmitochondria is created with expression cassettes for Cas9 and guide RNAthat are customized for expression in mitochondria. The plasmid DNA alsocontains donor DNA to facilitate site-specific genome editing. The Cas9gene is optimized for mitochondrial expression (SEQ ID NO: 24) and isoperably linked to a COX2 promoter and a terminator (SEQ ID NO: 25 and26, respectively). The optimization is performed by changing CTN codonsto TTA, GGG/GGC to GGT, GCG/GCC to GCT, CGG/CGC to CGT, CCG/CCC to CCT,AGC to AGT, AGG to AGA, ACG/ACC to ACT, TCG/TCC to TCT and GAG to GAA aswell as TGA stop codon to TAA. The polynucleotide encoding a guide RNAthat contains a variable targeting domain designed for the mitochondrial21S rRNA gene (SEQ ID NO: 27) is operably linked to a promoter andterminator for the expression of the mitochondrial 15S rRNA gene (SEQ IDNO: 28 and 29, respectively). The donor DNA fragment carries the 21SrRNA gene with the chloramphenicol resistance allele, C^(R)321. TheC^(R)321 mutation in the mitochondrial 21S rRNA gene can conferchloramphenicol resistance in yeast. For the selection of the plasmid inmitochondria, the plasmid can also carry a positive selectable markersuch as active 15S rRNA gene with the paromomycin resistance mutationdescribed above. This plasmid is transformed into mitochondria of yeastlines such as MCC123 [rho⁰] together with the other plasmid for nucleartransformation to select events of co-transformation of both plasmids inyeast. Transformed yeast cells are first colonized on media to allow theselection of nuclear transformants. By replica plating the colonizedcells on the plates spread with a yeast line carrying the oppositemating type and wild-type mitochondrial genome, the colonies that areresistant to chloramphenicol are identified through subsequentreplica-plating of mated cells on non-fermentable media such as YPGEwith chloramphenicol (4 mg/ml). The increased frequency ofchloramphenicol resistance colonies is confirmed by comparison with thefrequency of chloramphenicol resistance colonies produced by the plasmidwithout Cas9 and guide RNA. Successful genome editing is furtherconfirmed by sequencing of the edited site in mitochondrial DNA.

Example 5 Insertion of an Exogenous Gene into Mitochondrial DNA andElimination of Unmodified Mitochondrial DNA

In this example, similar to Example 4, mitochondria are transformed withan Edit Plasmid. The Edit Plasmid contains an element that allowsreplication in mitochondria, and additional components of asite-specific endonuclease system such as Cas9, guide RNA and donor DNA.The donor DNA is designed to be bounded by two regions homologous to themitochondrial genome for homologous recombination, which is facilitatedby site-specific DNA cleavages. Between the two homologous regions, theinsertion of an expression unit is demonstrated, consisting of a COXIIpromoter, a polynucleotide encoding GFP fluorescence protein and aterminator. The donor DNA can have multiple expression units with orwithout polycistronic expression; i.e., where multiple coding regionsare expressed under one promoter.

Two separate sites are targeted by Cas9-gRNA complexes in onedemonstration. One Cas9 cleavage site in the COB gene is designed(variable targeting domain of: TGTCCCATTAAGACATAAGGTACTTCTACA SEQ IDNO:30; which precedes a TGG PAM sequence), and another cleavage site inthe ATP9 gene (variable targeting domain of:TGGAGCAGGTATCTCAACAATTGGTTTATTAGGAGC SEQ ID NO:31; which precedes a AGGPAM sequence). One end of the donor DNA comprising polynucleotide coversthe COB cleavage site and the other end covers the ATP9 gene tofacilitate homologous recombination between the donor DNA andmitochondrial DNA. The donor DNA carries mutations in the sequence nearthe Cas9-gRNA cleavage sites to eliminate subsequent DNA cleavage afterhomologous recombination events. These mutations are designed to be“silent”; i.e., the mutated sequence has the same functionality as thewild type, such as replacement of one codon with a synonymous codonencoding the same amino acid. In addition to the modification at thecleavage sites, we also design Cas9-gRNA complexes that cleaveadditional sites between the two primary target sites in the wild-typemitochondrial DNA but not the donor DNA and the mitochondrial DNAproduced by homologous recombination of donor DNA. Additional cleavagesites facilitate the “Genome Sweep” action; i.e., elimination ofwild-type mitochondrial DNA without eliminating engineered mitochondrialDNA.

In a separate demonstration, the donor DNA contains a polynucleotideencoding lactoferrin in the place of GFP.

Example 6 Genome Editing of Mammalian Mitochondrial DNA

For Cas9 import into mammalian mitochondria, Cas9 protein withoutnuclear localization signal element is fused with a mitochondrialtargeting peptide. One such peptide is NDUFV2 MTS which has 32 aminoacid residues, NH2-MFFSAALRARAAGLTAHWGRHVRNLHKTVMQN—COOH (SEQ ID NO:32).In this case, the NDUFV2 signal sequence is fused with the aminoterminus of Cas9 to give a modified Cas9 (SEQ ID NO: 33). Alternatively,another signal peptide such as the one from citrate synthase(NH2-MALLTAAARLLGTKNASCLVLAARH—COOH; SEQ ID NO:34) that can function inhuman cells can be used to create a modified Cas9 (SEQ ID NO: 35). Apolynucleotide encoding a modified Cas9 gene (with a mitochondrialtarget sequence) is operably linked to a promoter element such as CMV byutilizing the human transfection vector, pSF-CMV-Amp, purchased fromSigma Aldrich or is operably linked to a inducible promoter such as theTET-inducible promoter of pTRE2hyg vector, which can be purchased fromClontech.

Similar to other examples, guide RNA is fused to a mitochondrialtargeting RNA; i.e., a sequence that allows import of RNA intomitochondria. In this experiment, RNAs that can be imported into humanmitochondria are used. One of them is the yeast tRNA^(Lys). The yeasttRNA^(Lys) and its variants can be imported into human mitochondria. Theother RNA used is 5S rRNA, which can be imported into humanmitochondria. In the latter case, the guide RNA is cloned into Loop Cthat can be dispensable for mitochondrial import (SEQ ID NO: 36).

In this experiment, the guide RNA is designed to target the COX3 gene(SEQ ID NO: 37). In the guide RNA, the variable targeting domain isfused with the tracrRNA sequence as well as with a mitochondrialtargeting RNA. The gRNA expression cassette consists of thepolynucleotide encoding the guide RNA operably linked to a promoter andterminator that are functional in human cells. In this example, the U6promoter for constitutive expression is used. For the 5S rRNA fusion,the promoter and terminator of the 5S rRNA gene (SEQ ID NO: 38) are alsoused. Guide RNA expression cassette is cloned into the plasmids carryingthe Cas9 expression cassettes or cloned into distinct transfectionvectors. Constructed plasmids are transfected into human cell lines suchas HeLa and HEK293 as well as HeLa and HepG2 Tet-Off cells for Cas9inducible expression from pTRE2hyg based constructs. Transfected cellsundergo selection in the presence of hygromycin. Preparation of cellculture and transfection are performed for inducible expression.

Cells are harvested three days after transfection and total DNA ofapproximately 106 cells is extracted using a DNA extraction kit. PCR isconducted to amplify the regions encompassing the target sites andamplified DNA is deep sequenced by use of a high-throughput sequencer(e.g., MiSeq Illumina sequencer). The sequence data are analyzed toconfirm modification at the target site.

Example 7 Genome Editing of Mammalian Mitochondrial DNA to ConferResistance to Chloramphenicol

In this example with mammalian cells, mitochondrial DNA is edited toconfer chloramphenicol resistance by a nucleotide substitution in the16S rRNA gene. For the purpose, three components, Cas9 protein, guideRNA and donor DNA, are targeted to mitochondria.

The chloramphenicol resistance in a mouse cell line can be mapped to asingle nucleotide change (CAP^(R)) in the mitochondrial 16S rRNA gene.The guide RNA is designed to include the CAP^(R) mutation site of thewild-type 16S rRNA gene. It is also designed in a manner that it willrecognize the wild-type sequence but not the donor DNA with the CAP^(R)mutation (SEQ ID NO: 39). The donor DNA is produced by PCR amplificationof the 16S rRNA region of the mouse CAP^(R) cells or is synthesizedartificially (SEQ ID NO: 40).

Cas9 and guide RNA are targeted to mitochondria as described in Example5. Plasmids with Cas9 and guide RNA expression cassettes are transfectedinto mouse cell lines such as NIH 3H3 as described above. The donor DNAis transformed into mitochondria. Transfected cells are cultured onmedia containing chloramphenicol (CAP). After the selection on CAP, theoccurrence of resistant cells through genome editing is confirmed incomparison with controls. Finally, 16S rRNA of the CAP^(R) cells issequenced to confirm genome editing at the molecular level.

Example 8 Introduction into Mammalian Mitochondria of Donor DNA andExpression Cassettes for Cas9 and Guide RNA

In this example, all components of genome editing including donor DNAare cloned in a plasmid DNA that is introduced into mammalianmitochondria. The plasmid DNA is introduced into mitochondria either ina circular form or in a linear form that has the ability to circularizein mitochondria. The plasmid DNA contains sequence that allows forautonomous replication in mitochondria. It can also encodes at least oneselectable marker to allow for selection after transformation intomitochondria. Such a selectable marker can be the active 16S rRNA genewith CAP^(R) mutation. The rep/ori and other elements for geneexpression in mitochondria present on the plasmid DNA may be derivedfrom species different from the target species for mitochondrial DNAediting. Additional DNA cleavage sites can be designed for the wild-typesequences that differ from the donor DNA as described in previousexamples.

Example 9 Introduction of Cas Endonuclease and Guide RNA into Plastids

To edit a chloroplast genome, Cas9 is modified to have a chloroplasttargeting amino acid sequence (also known as transit peptide, TP) at theN-terminus of the protein and to remove any nuclear localizationsignal(s). In addition, the nucleotide sequence of Cas9 iscodon-optimized for the plant species for optimum expression (SEQ ID NO:41 & 42; for nucleic acid and amino acid sequences, respectively). Thetransit peptides from chloroplast-targeted proteins such as ribulosebisphosphate carboxylase/oxygenase small subunit (rbcS), chlorophyll a/bbinding protein (Cab) and DnaJ8 are used in the experiments. Eachmodified Cas9 is engineered to have a transit peptide fusedtranslationally to the amino terminus of the Cas9 to create a TP-Cas9(SEQ ID NO: 46). Expression of a polynucleotide encoding such a fusionprotein is under control of a promoter functional in a plant, such as aCaMV 35S promoter. Cas9 without a transit peptide is used as a control(SEQ ID NO: 41 & 42).

For transport of a guide RNA into the chloroplast, RNA sequences areused that can import into the chloroplast. These plastid targeting RNAs(also referred to herein as “transit RNAs”), which can mediate import ofattached heterologous RNA, include vd-5′UTR (SEQ ID NO:48) and eIF4E1mRNA (SEQ ID NO: 49). Transcription of polynucleotides encoding thesefusion transcripts is under the control of a nuclear promoter functionalin a plant, such as the 35S CaMV promoter (e.g., 1.3-kb 35S promoter ofpBC-Yellow) or the U6 promoter; Chromosome 8 maize U6 polymerase IIIpromoter). Guide RNA without a plastid targeting RNA serves as a control(SEQ ID NO: 50).

As an alternative method of creating gRNAs, a sequence-specificendoribonuclease is used, such as Csy4 which is responsible forprocessing CRISPR transcript from Pseudomonas aeruginosa (SEQ ID NO:51-52, for nucleic acid and amino acid sequences, respectively). TheCsy4 recognition sequence is: 5′-GTTCACTGCCGTATAGGCAG-3′ (SEQ ID NO:53). Within the primary transcript, the gRNA sequence is flanked withCsy4 recognition sequences (SEQ ID NO: 54). A polynucleotide encodingthis sequence fused with a 5′ plastid targeting RNA is transcribed fromeither a 35S CaMV promoter or a U6 promoter in the nucleus and targetedinto the chloroplast. For targeting Csy4 protein into the chloroplast,one of chloroplast transit peptides listed in SEQ ID NO: 43-45 is used,as an N-terminal translational fusion to Csy4.

Example 10 Introduction into Plastids of RNA Encoding Both CasEndonuclease and Guide RNA

Plastid targeting RNA can transport heterologous RNAs into the plastid,which then are translated by the chloroplast translation machinery. Thischaracteristic is utilized to transport all the genome editingcomponents as RNA molecules into the chloroplast; transported mRNA issubsequently translated and the resulting proteins participate in theediting process. In this method, an expression cassette is madecomprising a promoter operably linked to a polynucleotide encoding anRNA comprising the following: plastid targeting RNA, rbs (ribosomebinding site), Cas9 coding sequence, rbs, Csy4 coding sequence, Csy4recognition sequence, gRNA, and Csy4 recognition sequence. Thisexpression cassette is integrated into the nuclear genome bytransformation. The promoter in the above recombinant DNA construct is apromoter functional in a plant, such as a CaMV 35S promoter. Theresulting RNA molecule is transported into chloroplast. Once it enterschloroplast, Cas9 and Csy4 proteins are produced by the chloroplasttranslation machinery. A complex of Cas9 and gRNA, which is processedfrom the transported RNA molecule by Csy4, finds and edits the targetsite in the chloroplast genome.

Example 11 Guide RNA Target Site Selection

Guide RNA target sites are selected from intergenic regions as well asgenic regions of the chloroplast genome. The latter examples includerpoB, psbA, rps15, and rp133. Deletion of the rpoB gene can show aphotosynthesis-defective phenotype. Deletion of the psbA gene can yielda photosystem II deficiency. Double deletion of rps15 and rp133 canresult in synthetic lethality under autotrophic conditions. Use ofweb-based Bioinformatics program, APE(http://biologylabs.utah.edu/jorgensen/wayned/ape/), facilitates theselection process for gRNA target sites.

To select gRNAs target sites for N. tabacum, the N. tabacum chloroplastgenome sequences are used. For gRNAs target sites for N. benthamiana,either public sequence deposition or direct sequencing of target regionsin N. benthamiana chloroplast genome is used, as the total chloroplastgenome sequence of N. benthamiana is not available. In addition, N.tabacum chloroplast DNA sequence is also used for the design of gRNAtarget sites for N. benthamiana since closely related plant species canhave highly conserved chloroplast DNA sequences. Similarly, chloroplastGlycine max (strain: William 82) genomic sequence from Organelle GenomeResources at NCBI is used as a reference genome for designing tentativegRNA target sites in soybean chloroplast DNA, pending sequencing of thespecific line that is transformed.

For editing of the indicated genic sequence regions, the followingsequences are selected for variable targeting domains. The term “Nt”corresponds to “Nicotiana tabacum”, the term “Cp” corresponds to“Chloroplast” and the term “Glma” corresponds to “Glycine max”. When thevariable targeting domain is on the reverse complement of the genicsequence, the term “reverse” is indicated.

For NtCp_rpoB (RNA polymerase beta chain) (SEQ ID NO: 55) 1.(SEQ ID NO: 56) TTAGAGGAAGAGCCAAACAG 2. (SEQ ID NO: 57)CTTGCTATAGCCGAACGCGA For NtCp_psbA (photosystem II proteinD1) (SEQ ID NO: 58) 1. (SEQ ID NO: 59) GTTGATGAATGGTTATACAA 2.(SEQ ID NO: 60) GATGATCCCTACCTTATTGA For NtCp_rps15 (ribosomal proteinS15) (SEQ ID NO: 61) 1. (SEQ ID NO: 62) ATTTCTCAAGAAGAAAAGAG 2.(SEQ ID NO: 63) TCAATTTCACCAATAAGATAFor NtCp_rpl33 (50S ribosomal protein L33) (SEQ ID NO: 64) 1.(SEQ ID NO: 65) GATATATTACTCAAAAGAAC 2. (SEQ ID NO: 66)AGTGTTGATAAGGTATCAAG For GlmaCp rpoB (RNA polymerase betachain) (SEQ ID NO: 67) 1. (SEQ ID NO: 68) TGTCTAAAACTACCTACAGG 2.(SEQ ID NO: 69) AGCGGAATTTCGGTCTATAC (reverse)For GlmaCp psbA (photosystem II protein D1) (SEQ ID NO: 70) 1.(SEQ ID NO: 71) GGTGTAGCTGGTGTATTCGG 2. (SEQ ID NO: 72)TCTAGATCTAGCTGCGATCG (reverse) For GlmaCp_rps15 (ribosomal proteinS15) (SEQ ID NO: 73) 1. (SEQ ID NO: 74) ATAGAATACGAAGACTTACT (reverse)2. (SEQ ID NO: 75) TGTCAAAGAAAGATAGAATA For GlmaCp_rpl33 (50S ribosomalprotein L33) (SEQ ID NO: 76) 1. (SEQ ID NO: 77) CGTTGTTGCAAACATACAAT(reverse) 2. (SEQ ID NO: 78) ACAGAATACGCCTAGTCGATFor Nicotiana benthamiana rps16(ribosomal protein S16) (SEQ ID NO: 79) 1. (SEQ ID NO: 80)TTGTGGATTTGTACATCCAC (reverse) 2. (SEQ ID NO: 81) TTGAACTGTTTGAAAGTTAT(reverse) For Nicotiana benthamiana matK (maturase K) (SEQ ID NO: 82) 1.(SEQ ID NO: 83) CTTGTGCTAGAACTTTAGCT 2. (SEQ ID NO: 84)CGTTCATCTGGAAATCTTGG (reverse)

For editing of the intergenic regions, the following sequences areselected for variable targeting domains.

Nicotiana tabacum: 1. (NtChrC;57408..57389) (SEQ ID NO: 85)AAGAACTTCCCCCTTGACAG 2. (NtChrC;59412..59393) (SEQ ID NO: 86)TATACAGGATGGGTAGAAAG 3. (NtChrC;59622..59603) (SEQ ID NO: 87)ATATAATTTTTAATAAAGGG 4. (NtChrC;65704..65723) (SEQ ID NO: 88)CTAGTCTTCGACACAAGAAA Glycine max: 1. (GlmaCp_NC_007942.159039-59058)(SEQ ID NO: 89) ATAACAGAAGTTAAAGAAGA 2. (GlmaCp_NC_007942.159100-59119)(SEQ ID NO: 90) ATCTGGAAACCATAGAACAG 3. (GlmaCp_NC_007942.162057-62038)(SEQ ID NO: 91) CTATTTCGACACAAACAAGA 4. (GlmaCp_NC_007942.162361-62380)(SEQ ID NO: 92) CTTTCTTTGACGAATTCGAG

Example 12 Transformation with Polynucleotides Encoding Cas Endonuleaseand Guide RNAs

Gene cassettes encoding (a) Cas9 fused to a transit peptide; and (b)gRNA fused with vd-5′UTR or eIF4E1 mRNA as described above are subclonedinto a binary vector, such as pPZP and introduced into plants either fortransient or for stable expression. DNA encoding Csy4 fused to a transitpeptide is also transformed into plants in some experiments. Any ofseveral methods may be used to transform plants with DNA sequences.These include agroinfiltration, biolistic bombardment, and floral dipmethod.

Similar approaches are also applicable for other plant species includingdicots such as canola and monocots such as rice, wheat and corn.

Example 13 Introduction of Donor DNA into the Plastid Via ReverseTranscriptase

A donor DNA is introduced into the plastid genome to edit the genome inat least one way selected from the group consisting of: (1) creation ofa point mutation in a target gene; (2) replacement of an endogenouscoding region or regulatory sequence with a heterologous DNA sequence;and (3) insertion of a heterologous DNA sequence (e.g., for expressionof a heterologous protein or RNA; for regulation of an endogenous gene).

In above examples several methods are presented for delivery of Cas9 andgRNAs into a chloroplast. In the current example, a donor DNA is alsodelivered into a chloroplast. In one method, a donor DNA for homologousrecombination in a chloroplast is generated through reversetranscription of an RNA donor molecule which is transported into achloroplast by transit RNA-guided transport. The RNA donor molecule,which is transcribed from transformed nuclear genome, contains thefollowing: (1) a transit RNA, (2) sequences for homologousrecombination; (3) a polynucleotide modification template sequencehaving at least one of the following: an endogenous sequence with anintended mutation (e.g., a site-specific mutation in the 16S rRNA) and aheterologous sequence (e.g., a heterologous protein coding sequence);and (4) a sequence that serve as a priming site for reversetranscriptase. In the homologous DNA regions, additional mutations,e.g., silent point mutations, are introduced into the sequence todistinguish these regions from additional gRNA target sites on thechloroplast DNA. The additional gRNA target sites are used to cleavenon-transformed copies of chloroplast DNA. Reverse transcriptase proteinis targeted into the chloroplast through a translational fusion with anyof plastid targeting peptides described in SEQ ID NO: 43-45.Alternatively, an mRNA molecule (with a plastid rbs) encoding a reversetranscriptase is transported into the chloroplast as a fusion moleculewith any one of plastid targeting RNAs described in SEQ ID NO: 48-49 andtranslated in chloroplast by the endogenous translation machinery.

Example 14 Introduction of Donor DNA into the Plastid Via Co-Bombardmentwith Two Polynucleotides

Another method to deliver donor DNA in conjunction with Cas9 and gRNAsis achieved through co-bombardment of two DNA molecules. In thisapproach, a first DNA molecule encoding Cas9 and gRNAs (employingchloroplast transport methods as described in previous examples) istargeted for transformation into the nuclear genome. A second DNAmolecule, having a donor DNA sequence and homologous recombinationsequences, is targeted for transformation into the chloroplast genome.The second DNA molecule also can contain a chloroplast origin ofreplication. For transformation both DNA molecules are delivered toplant cells by biolistic bombardment. Biolistic particles are preparedas follows: (1) particles are coated with both DNA molecules eithersimultaneously or sequentially; or (2) particles are separately coatedwith each DNA molecule and then combined with the same molar ratio. Forselection of nuclear transformation, commonly used antibiotic markers,such as nptII and bar, and/or fluorescent protein markers can beemployed. For selection of chloroplast transformation, antibioticmarkers such as aadA and/or fluorescent protein markers are used. Theexpression cassette for the chloroplast transformation selectable markeris either part of the donor DNA carrying polynuclotide that isintegrated into the plastid genome or is placed outside of the donor DNAregion, but remains on the delivered DNA molecule without beingintegrated into the chloroplast genome.

In a variation of above example of polynucleotide modification templatedelivery into the chloroplast, polynucleotides encoding Cas9 and gRNA(with or without Csy4) are transformed into the nuclear genome first.Gene expression of these components are under the control of induciblepromoters. With the aid of selection markers (antibiotic markers and/orfluorescent marker proteins) stably transformed plants are selected. Asecond transformation is performed to transform chloroplast DNA with aDNA molecule containing a polynucleotide modification template DNA,homologous recombination sequences and a selectable marker such as aadAand/or a fluorescent marker protein. Selection of transformants isperformed in the presence of selection agents for both nuclear andchloroplast transgenes and under conditions where the inducible promoteron the nuclear transgenes is active to transcribe Cas9 and gRNAs, whichare subsequently transported into the chloroplast via the mechanismdescribed in the previous examples.

Example 15 Introduction of Donor DNA into the Plastid ViaAgrobacterium-Mediated Transformation

Donor DNA transport into the chloroplast is also performed viaAgrobacterium-mediated transformation. A stable transgenic line whichcontains polynucleotides encoding Cas9 and gRNAs with an induciblepromoter is created, as described above. This line is then transformedwith a modified Agrobacterium strain, wherein the modification comprisesthe following: (1) addition of a chloroplast transit peptide fused toVirD2; (2) deletion of VirE2; and (3) removal of nuclear localizationsignals from VirD2. A binary vector is constructed having apolynucleotide modification template, homologous recombination sequencesand a selection marker such as aadA and/or a fluorescent marker proteinin between right and left T-DNA borders and transformed intoAgrobacteria. For transformation, stable transgenic lines withpolynucleotides encoding Cas9 and gRNAs are incubated with Agrobacteria.VirD2 protein which is covalently linked to single-stranded T-DNA entersinto plant cells and is transported into the chloroplast via theN-terminal transit peptide. Transgenic selection is imposed by dualselection with nuclear (nptII) and chloroplast (aadA) markers and underconditions where the inducible promoter is active to transcribepolynucleotides encoding Cas9 and gRNAs, which are subsequentlytransported into chloroplast by the mechanism described in the previousexamples.

Example 16 Introduction into Plastids of Donor DNA and ExpressionCassettes for Cas9 and Guide RNA

In this example, a DNA plasmid (“Edit Plasmid”) that can replicate inplastids and encodes components of a site-specific endonuclease systemsuch as Cas9, guide RNA and donor DNA, is directly introduced into theplastid. The delivery of nucleic acids and proteins can be accomplishedby use of methods such as bombardment (biolistics), electroporation andother available methods. Here an example in tobacco chloroplasts isshown.

The Edit Plasmid for tobacco chloroplasts is constructed as follows.Polynucleotides encoding Cas9 and guide RNA are cloned into the vectorand are operably linked to appropriate promoters and terminators toallow for expression in tobacco chloroplasts. Alternatively, these twocoding regions may be linked and transcribed polycistronically under onepromoter. The polycistronic RNA may be processed to give rise toseparate functional RNA molecules for genome editing, one for Cas9translation and the other for guide RNA. A polynucleotide encoding aselectable marker that enables selection of the plasmid in chloroplasts,such as the aadA gene conferring spectinomycin resistance, is alsopresent on the plasmid DNA, operably linked with an appropriate promoterand a terminator active in chloroplasts. An expression cassette encodinga negative selectable marker gene is also present on the plasmid toallow for counter selection, i.e., selection of chloroplasts withoutEdit Plasmid after editing and subsequent elimination of wild-typechloroplast DNA has been achieved. The dao gene is one such negativeselectable marker gene. Furthermore, an element that allows forreplication of the Edit Plasmid is also present in the vector. Such anelement can be derived from the chloroplast DNA of the target species oralternatively from chloroplast DNA of another species, as well as fromcompletely synthetic sources. In addition, donor DNA is present on thevector to allow for precise DNA editing and/or the precise insertion ofheterologous DNA elements at specific sites in the chloroplast DNA.

As one example, the wild-type psbA gene in tobacco chloroplast DNA isreplaced with an allele carrying a single nucleotide substitution thatconfers resistance to the herbicide triazine. Such a mutation can bepresent in herbicide tolerant plants in nature. For DNA cleavage in thevicinity of the mutation site, guide RNA to target the following DNAsequence is designed.

(SEQ ID NO: 93) ACGAGAGTTGTTGAAACTAGCATATTGGAAGATCAA

The PAM sequence (TGG) is in bold font.

The donor DNA contains the following sequence with five mutations shownin bold font.

(SEQ ID NO: 94) ACGAGAGTTATTGAAT G TAGCATACTGAAAGATCAA

The atrazine resistance mutation (G) is underlined. The four additionalchanges that do not alter protein sequence are present to eliminate thedonor DNA as being a target for the guide RNA designed for theendogenous wild-type psbA sequence. In particular, one change eliminatesthe PAM sequence critical for guide RNA pairing to the targetpolynucleic acid (e.g., target DNA) sites.

To facilitate homologous recombination, the donor DNA is bounded bylonger homologous sequences upstream and downstream of the abovesequence.

The Edit Plasmid is transformed into tobacco chloroplast by thebiolistic approach as described in Chloroplast Biotechnology Methods andProtocols, Pal Maliga (Editor), Methods in Molecular Biology, Springer,New York (2014) (Cells with transformed chloroplasts are selected on themedia containing spectinomycin. After the cultivation of callus cells onthe selective media, calli are transferred to the media containingatrazine to assess the frequency of site-specific genome editing withthe donor DNA. Sequencing of callus cells resistant to the herbicideconfirms the successful genome editing at the molecular level.

To increase the rate of obtaining homoplasmic chloroplasts withengineered DNA, additional target sites are designed in the wild-typesequence covered by the corresponding homologous regions adjacent to thedonor DNA. To protect the donor DNA and edited DNA in the chloroplast,donor DNA harbors silent mutations that avoid cleavage by Cas9endonuclease; e.g., replacing codons with synonymous codons coding forthe same amino acids. Expression cassettes encoding the gRNA(s)corresponding to those additional target sites are cloned into the EditPlasmid vector for expression in chloroplasts. The donor DNA with theadditional gRNA target sites mutated (for protection from Cas9endonuclease) is also present in the Edit Plasmid.

The above Edit Plasmid with increased Genome Sweep activity istransformed into tobacco chloroplast as described above. Cells withtransformed chloroplasts are selected on the media containingspectinomycin. After the cultivation of callus cells on the selectivemedia, calli are transferred to the media containing atrazine to assessthe frequency of site-specific genome editing with the template DNA.Sequencing of callus cells resistant to the herbicide confirms thesuccessful genome editing at the molecular level.

When stable inheritance of edited organellar DNA is achieved, the EditPlasmid can be segregated out in progeny plants under non-selectiveconditions for the Edit Plasmid. The segregation process can befacilitated by utilizing the negative selectable marker encoded in theEdit Plasmid, e.g., D-valine selection for the dao gene.

Example 17 Regulatory Elements for Plastid Gene Expression

Expression cassettes may be constructed that have a promoter functionalin a plastid operably linked to either: (a) a donor polynucleotide; or(b) a plurality of donor polynucleotide arranged as a polycistronicunit. A desired 5′-UTR can also be present in the expression cassette,operably linked to the 3′-end of the promoter.

In one expression cassette, the polynucleotide (or polynucleotides) tobe transcribed can be operably linked to the following promoter::5′-UTRregulatory elements:

-   -   (a) the maize clpP promoter in combination with the maize clpP        5′-UTR;    -   (b) the maize clpP promoter in combination with the 5′ UTR from        gene 10 of bacteriophage T7;    -   (c) the tomato psbA promoter in combination with the T7g10        5′-UTR; and    -   (d) the tomato rm16 promoter in combination with the accD-mod        5′-UTR.

The above regulatory elements can be obtained by PCR amplification.

Example 18 Pest Resistance Genes for Expression in Organelles

An expression cassette for use in organelle transformation isconstructed using the wild-type nucleic acid sequence from Bacillusthuringiensis serovar kurstaki (U89872; SEQ ID NO:108) encoding thefull-length native HD73 Cry1Ac delta-endotoxin (SEQ ID NO:109).Alternatively, a truncated native nucleic acid sequence (SEQ ID NO:110)is used, which encodes the active truncated Cry1Ac fragment.Additionally, in some cases, the nucleic acid sequence encoding thefull-length or truncated Cry1Ac protein is codon-optimized for theorganelle of interest.

In some cases, additional polynucleotides that encode proteins useful inconferring insect resistance to a plant are included in the aboveexpression cassette as a polycistronic unit, or are expressed fromseparate expression cassettes. These polynucleotides encode thefollowing: (a) the Cyt1Aa protein from Bacillus thuringiensis serovarisraelensis (Gene ID: 5759908; SEQ ID NO:111); (b) the 20 kDa accessoryprotein from Bacillus thuringiensis serovar israelensis (pBt024; SEQ IDNO:112); and (c) the 19 kDa accessory protein from Bacillusthuringiensis serovar israelensis, (pBt022; SEQ ID NO:113).

Example 19 Engineered Plant with Increased Pest Resistance

In this example, a plant (e.g., soybean plant) is engineered withincreased resistance to pests. Optionally, the plant also has increasedresistance to herbicides.

The site-specific endonuclease system (e.g., Cas9, guide RNA, and donorDNA) of the disclosure is used to introduce one or more pesticidalproteins into the organellar (e.g., plastid) genome of a plant cell(e.g., soybean cell). The one or more pesticidal proteins or theirfragments are selected from the group consisting of: Cry1Ac, Cyt1Aa(e.g., SEQ ID NO:109 or SEQ ID NO:110), Cry1Ab, Cry2Aa, Cry1I, Cry1C,Cry1D, Cry1E, Cry1Be, Cry1Fa and Vip3A.

In some cases, one or more accessory proteins are also introduced intothe organellar (e.g., plastid) genome of the plant cell. The one or moreaccessory proteins can bind to a pesticidal protein and are selectedfrom the group consisting of: a 20 kDa accessory protein and a 19 kDaaccessory protein.

Additionally or independently, in some cases, the site-specificendonuclease system (e.g., Cas9, guide RNA, and donor DNA) is used tointroduce one or more heterologous donor polynucleotides encoding adsRNA, a siRNA, and/or a miRNA, wherein the dsRNA, the siRNA and themiRNA can suppress at least one target gene present in a plant pest,into the organellar (e.g., plastid) genome of the plant cell (e.g., thesoybean cell). The dsRNA, the siRNA and the miRNA can suppress at leastone target gene selected from the group consisting of: proteasome A-typesubunit peptide (Pas-4), ACT, SHR, EPIC2B and PnPMAI. The RNAinterference-based mechanism can be used to protect the engineeredplants from pests.

Optionally, in some cases, one or more herbicide tolerance proteins isalso introduced into the organellar (e.g., plastid) genome of the plantcell using the site-specific endonuclease system (e.g., Cas9, guide RNA,and donor DNA) of the disclosure. The herbicide tolerance protein can beat least one selected from the group consisting of: a4-hydroxphenylpyruvate dioxygenase (HPPD), a sulfonylurea-tolerantacetolactate synthase (ALS), an imidazolinone-tolerant acetolactatesynthase (ALS), a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a glyphosate-tolerant glyphosate oxidoreductase (GOX),a glyphosate N-acetyltransferase (GAT), a phosphinothricin acetyltransferase (PAT), a protoporphyrinogen oxidase (PROTOX), an auxinenzyme or receptor, a P450 polypeptide and an acetyl coenzyme Acarboxylase (ACCase).

Example 20 Genetic Modification of Yeast Mitochondrial DNA by the EditPlasmid Approach

To show mitochondrial genome editing with our methodology in yeast,Saccharomyces cerevisae, various Edit Plasmid constructs were designed.The reference sequence used was a compete mitochondrial genome sequencefrom the Saccharomyces Genome Database (SGD),https://www.yeastgenome.org/. The targeted gene was the COX1 gene (alsocalled oxi3 gene). Mutants of this gene previously have been shown tohave a respiration-defective phenotype(https://www.yeastgenome.org/locus/S000007260). The following four guideRNA target sites in the COX1 gene were used (when the targeting sequencewas on the reverse complement of the genic sequence, the term “reverse”is indicated):

1) (SEQ ID NO: 116) TTCTTTGAAGTATCAGGAGGTGG; 2) (SEQ ID NO: 117)ATGATTATTGCAATTCCAACAGG; 3) (SEQ ID NO: 118) GCTATTTTTAGTGGTATGGCAGG;and 4) (SEQ ID NO: 119) ACCATGTAAATATTGTGAACCAGG. (reverse)

The last three nucleotides in each sequence correspond to the PAMsequence. The first target site resided in exon 5, the second in exon 4,the third one in exon 1 and the forth one at the junction of 3′ end ofexon 1 of the mitochondrial COX1 gene. Each Edit Plasmid contained aguide RNA expression cassette encoding guide RNA(s) directed to eitherone or two of the four COX1 target sites. The variable targeting domainof each guide RNA did not contain the 3-nucleotide PAM sequence listedabove.

Yeast mitochondrial transformation was performed by following theprotocol developed by the Fox lab (Fox et al. 1988 Proc Natl Acad SciUSA 85:7288-7292; Bonnefoy and Fox 2001 Methods Enzymol 350:97-111). Itpreviously has been shown that plasmids derived from pBR322 were capableof replicating in yeast mitochondria (Fox et al. 1988). One of theplasmids derived from pBR322, pHD6 was used, and the plasmids had beensuccessfully transformed into yeast mitochondria in the past(Green-Willms et al. 2001 J Biol Chem 276: 6392-6397). All clonedfragments of pHD6 by digesting with PstI and HindIII are deleted exceptthe genomic fragment of COX3 gene to leave the pBR322 backbone forcreating our constructs. The COX3 fragment (0.75 kb PacI-MboI) was usedas a screenable marker for mitochondrial transformants with itscapability to rescue the cox3 deletion mutant cox3-10 as described inFox et al., 1988. The Edit Plasmid constructs contained the followingelements in the pBR322 backbone: Cas9 expression cassette, guide RNAexpression cassette and donor DNA in the case of DNA replacementexperiments. The Cas9 expression cassette had a Cas9 coding sequencethat was optimized for the expression in yeast mitochondria (SEQ ID NO:120). As part of codon optimization, the Cas9 codons that were not usedat all or were rarely used in yeast mitochondria were replaced withcodons that were used frequently. Also, a number of tryptophan codonswere replaced with TGA, which is a stop codon in the universal codontable but is translated into tryptophan in yeast mitochondria (Fox 1979Proc Natl Acad Sci USA 76: 6534-6538). This was designed to preventexpression of Cas9 in the nucleus after microprojectile DNAtransformation. The expression cassette with the optimized Cas9 ORF wassynthesized with the minimal promoter with 5′ UTR and terminator of theCOX2 gene; these regulatory elements were flanked with PstI and HindIIIsites, respectively (SEQ ID NO: 121 and SEQ ID NO: 122). The minimalpromoter and terminator, which had the length of 71 and 119 bp,respectively (Mireau et al. 2003 Mol Gen Genomics 270:1-8), were chosenwith the purpose of suppressing homologous recombination at the sitesand avoiding integration into the mitochondrial genome. Several uniquerestriction sites (XbaI, NotI and NcoI sites) were included at theHindIII end to facilitate cloning of additional elements. One suchelement was the guide RNA expression cassette. Guide RNAs targeting theCOX1 sequences described above were created by fusion of each targetingsequence with the tracrRNA sequence (SEQ ID NO: 123). Each guide RNAexpression cassette encoded either one or two guide RNAs, which weredirected to the corresponding one or two of the four COX1 target sites.

The guide RNA expression cassette contained the following elements in 5′to 3′ orientation: a minimal COX3 promoter (SEQ ID NO: 124); a tRNAgene, tF(GAA) (SEQ ID NO: 125); a single guide RNA directed to a COX1site; a second tRNA gene, tW(UCA) (SEQ ID NO: 126); and a minimal COX3terminator element (SEQ ID NO: 127). The constructs with two guide RNAswere created by combining guide RNAs directed to COX1 sites 1 and 2, aswell as to sites 3 and 4. When two guide RNA encoding sequences werepresent, the second one was fused directly after the tW(UCA) sequenceand was flanked by a third tRNA gene, tM(CAU) (SEQ ID NO: 128) at the 3′end and before the COX3 terminator. The guide RNA expression cassetteswith promoter and terminator elements were synthesized with a NotI siteat the 5′ end and a NcoI site at the 3′ end to allow directional cloninginto the pBR322 backbone that carries the Cas9 expression cassette.

For the DNA replacement experiments, the donor DNA carrying the GFP genewas synthesized and cloned into the NcoI site of constructs that encodedtwo guide RNAs. The nucleotide sequence (SEQ ID NO: 129) encoding GFPwas codon optimized for expression in yeast mitochondria as done forCas9 (see above). Several codons for tryptophan were changed to TGA,assuring GFP expression only in mitochondria. Also, the GFP codingregion was designed to be in frame with the COX1 gene after DNAreplacement. Both ends of the GFP ORF were fused with the COX1 genomicsequences at the external junction of the Cas9 cleavage sites. HR1-HR4correspond to four short homology regions used in construction of theEdit Plasmids; they were each immediately adjacent to the correspondingguide RNA target site. The length of the homologous region at each endwas chosen to be relatively short to minimize endogenous homologousrecombination without Cas9 cleavages, i.e. 144 bp adjacent to the #1guide RNA site (HR1; SEQ ID NO: 130), 115 bp adjacent to the #2 guideRNA site (HR2; SEQ ID NO: 131), 64 bp adjacent to the #3 guide RNA site(HR3; SEQ ID NO: 132) and 93 bp adjacent to the #4 guide RNA site (HR4;SEQ ID NO; 133). This design should facilitate DNA replacement inducedby Cas9 activity and not by general homologous recombination.Additionally, the Edit Plasmids should remain autonomous withoutintegrating into the genome. Furthermore, sequence variations wereincluded at the guide RNA recognition sites within the donor DNA, sothat the mitochondrial DNA after replacement would no longer berecognized by the guide RNA/Cas9 complex. This was done to prevent thedeletion of the replaced DNA from the gene-edited mitochondrial genome.The variant of the first target site is listed under SEQ ID NO: 134,where 7 of the 20 nucleotides in the guide RNA recognition site havebeen changed. The variant of the second site was created by deleting 16nucleotides at the 5′ end of the recognition site (SEQ ID NO: 135). Thethird target site was modified by deleting the last five nucleotides(SEQ ID NO: 136). The fourth target site was modified by deleting 14nucleotides at the 5′ end (SEQ ID NO: 137).

The constructs made for this experiment are presented in Table 1.

TABLE 1 Components of Edit Plasmids for Yeast Mitochondria ConstructExpr Cassette 1* Expr Cassette 2** Donor DNA HS1 Cas9m tF:sgRNA-3:tW N/AHS2 Cas9m tF:sgRNA-4:tW N/A HS3 Cas9m tF:sgRNA-3:tW:sgRNA-4:tM N/A HS4Cas9m tF:sgRNA-3:tW:sgRNA-4:tM HR3:GFPm:HR4 HS5 Cas9mtF:sgRNA-3:tW:sgRNA-4:tM HR3:GFPm:HR4*** HS6 N/AtF:sgRNA-2:tW:sgRNA-1:tM HR1:GFPm:HR2 HS7 N/A tF:sgRNA-3:tW:sgRNA-4:tMHR3:GFPm:HR4 HS8 Cas9m tF:sgRNA-2:tW:sgRNA-1:tM HR1:GFPm:HR2 HS9 Cas9mtF:sgRNA-2:tW:sgRNA-1:tM N/A HS10 Cas9m tF:sgRNA-1:tW N/A HS11 Cas9mtF:sgRNA-2:tW N/A *Each Expression Cassette 1 had the COX2 promoter with5′ UTR and the COX2 terminator. **Each Expression Cassette 2 had theCOX3 promoter and the COX3 terminator. ***The Donor DNA is in reverseorientation with respect to the construct HS4.

The constructs created were transformed into yeast lines that lackedmitochondrial DNA (rho⁰), MCC109rho0 (MATα ade2 ura3 kar 1), using thebiolistic microprojectile method as described in Bonnefoy and Fox, 2001.The transformation was performed together with pYES2 as a carrierplasmid with URA3 selectable marker, so that URA⁺ nuclear transformantscould be selected first on minimal medium lacking uracil in supplements.To identify mitochondrial transformants, URA⁺ colonies were assayed forthe ability of rescuing a cox3 deletion mutant through a cross withMCC125 (MATa lys2 rho⁺ cox3-10). The assay was repeated at least twiceto obtain clean colonies with Edit Plasmids in the mitochondria.Isolated lines containing Edit Plasmids were then crossed with linescontaining the wild-type mitochondrial genome, CUY563 (MATa ura3 ade2leu2 ade3 rho⁺) and NB80 (MATa lys2 arg8 ura3 leu2 rho⁺), to analyze thegenome editing effect by Cas9 at the target sites. In nuclearchromosomes subjected to double-strand breaks, one might expect a highfrequency of mutations such as small deletions or insertions at thetarget sites. They are the results of Non-Homologous End-Joining (NHEJ)repair at the site of DNA cleavage triggered by the guide RNA dependentCas9 activity. In yeast, 90% of the repair of double-strand breaks inchromosomes occurs by homologous recombination (Ricchetti et al. 1999Nature 402:96-100). In mitochondria, where multiple copies ofmitochondrial DNA are present in one organelle, the repair of dsDNAbreaks through homologous recombination is expected to be significantlymore frequent than in the nucleus. Under this circumstance, thefrequency of indel mutations caused by re-ligation of DNA ends isexpected to be extremely low in mitochondria. Due to this consideration,we focused on the detection of events caused by repair throughhomologous recombination, i.e., replacement with artificial donor DNA.

To assay for DNA replacement through Cas9 induced cleavages, theconstruct HS8 and its control construct HS6 were each transformed into astrain lacking mitochondrial DNA as described above. Each constructcarried the donor DNA with GFP as well as two corresponding guide RNAgenes (#1 and #2) but HS6 lacked the Cas9 expression cassette. Linesthat contained each construct were identified by subsequent screeningfor their capability of rescuing the cox3 deletion mutant. The isolatedmitochondrial transformants then were crossed with strains carrying thewild-type mitochondrial genome, CUY563 and NB80, to observe the effectof Edit Plasmids on the mitochondrial genomic DNA. The DNA replacementevents at the cleavage sites then were assayed by PCR amplification ofpooled cells two days after the crosses. Primer sets were used whereinone primer was from the mitochondrial genomic region in the vicinity ofthe cleavage sites and the other primer was from the donor DNA region,selected so that the desired PCR product could only be amplified from acorrectly replaced DNA in the mitochondrial genome but not from thewild-type mitochondrial DNA nor from the Edit Plasmid. The followingfour primer pairs were used: primers C and 12 for the 5′ end junction;and for the 3′ end junction, primers D and 11, E and 11, and F and 11.Primers C, D, E and F were specific to the genomic region of the COX1gene (SEQ ID NO: 138, 139, 140 and 141, respectively). Primers 11 and 12were specific to the GFP gene (SEQ ID NO: 142 and 143, respectively).The PCR amplification was performed as follows: Step 1: 94° C. for 7min, step 2: 94° C. for 30 sec, step 3: 52° C. for 30 sec, step 4: 60°C. for 1 min 30 sec, step 5: go to step 2 for 39 times, step 6: 60° C.for 10 min. The low temperature for the extension reaction was chosen toaccommodate AT-rich genomic sequences. After PCR amplification, weobserved the expected size of the DNA fragments from each end of thereplaced DNA by using the above four distinct pairs of primers. Nocorresponding DNA fragments were amplified in the cell samples that werecrossed with the line carrying the control construct.

The amplified DNA fragments were sequenced directly. FIG. 1 presents thesequence (SEQ ID NO: 144) obtained from PCR amplification of thereplaced DNA locus in transformed yeast mitochondrial DNA modified bythe Edit Plasmid approach. Underlined sequences at the 5′ and 3′ endsindicate wild-type mitochondrial genomic sequences that are not presenton the Edit Plasmid. Sequences in bold font indicate the shorthomologous regions present in the donor DNA and adjacent to thecorresponding guide RNA target sites. Sequences that have doubleunderlining indicate the modified guide RNA target sites present in thedonor DNA; altered nucleotides are shown in bold font. The guide RNAtarget sites in the replaced DNA have been modified to prevent nucleaseactivity after integration into the mitochondrial genome. Thecodon-optimized GFP coding region is presented in italics. Sequencespresented in lower case correspond to primers C and F that were used foramplification of the replaced DNA locus. Homologous recombinationoccurred as expected; i.e., there were no sequence changes either in thereplaced DNA or in the surrounding wild-type mitochondrial DNA.

The sequence (SEQ ID NO: 144; FIG. 1) covering the replaced regionmatched with the construct completely. Also shown in FIG. 1 aresequences at the 5′ and 3′ ends (shown with underlining) that arewild-type mitochondrial genomic sequences not present on the EditPlasmid, which are contiguous to the HR regions (shown in bold font)present in the Edit Plasmid. In summary, DNA replacement was observed inyeast mitochondria by use of an Edit Plasmid that encodes a Cas9expression cassette, a multiple guide RNA expression cassette and adonor DNA template.

Furthermore, single colonies were isolated from the cross between theHS8 line carrying the Edit Plasmid and wild-type strain, NB80. GFPsignal was confirmed from a fraction of colonies when viewed through afluorescence microscope.

In order to show the autonomously replicating nature of the EditPlasmids in mitochondria, we attempted the rescue of plasmids from thecells after the crosses described above. 1 ml of overnight cell cultureafter each cross was sampled and subjected to the total DNA isolation.200 ng of total DNA obtained by use of the Quick-DNA Miniprep Plus Kit(Zymo Research) were digested with ApaI and SphI to cleave pYES2 plasmidDNA in the total DNA fraction; the HS8 plasmid should remain intact asit doesn't possess these restriction sites. After inactivating therestriction enzymes at 65° C. for 20 min, the DNA was used to transformE. coli cells. Multiple colonies that grew on LB medium containingcarbenicillin were identified. DNA was isolated, subjected to digestionwith several restriction enzymes, and the digestion products wereseparated by gel electrophoresis. A number of plasmids were identifiedfrom two independent crosses that showed a digestion pattern identicalto the original HS8 construct, demonstrating that rescue of the originalEdit Plasmid HS8 was successful. This showed that Edit Plasmids remainedas autonomously replicating DNA in the presence of wild-typemitochondrial DNA, not integrated into the organelle genome.

Example 21 Genetic Modification of Chlamydomonas reinhardtii ChloroplastDNA by the Edit Plasmid Approach

Guide RNA target sites were selected from genic regions of theChlamydomonas reinhardtii chloroplast genome. The reference sequenceused was a compete chloroplast genome sequence from NCBI (Accessionnumber: NC_005353 and Version number: NC_005353.1). The targeted genewas psaA. Mutants of this gene previously have been shown to have aphotosynthesis-defective phenotype (Redding et al. 1999, J Biol. Chem.274: 10466-10473). To help design and select guide RNA target sites, aweb-based Bioinformatics program was employed—CRISPOR(http://crispor.tefor.net/, Haeussler et al. 2016 Genome Biology17:148-159). The following sequences were selected as guide RNAtargeting sites for editing of exon 3 in the psaA gene. When thetargeting sequence was on the reverse complement of the genic sequence,the term “reverse” is indicated. For each 23 nucleotide target sitelisted below, the first 20 nucleotides are the targeting sequencepresent in each corresponding guide RNA and the last 3 nucleotides arethe PAM sequence.

1. (SEQ ID NO: 145) GGTTTAAACCCTGTTACTGGTGG 2. (SEQ ID NO: 146)CTTCACCTGTAAATGGACCACGG (reverse) 3. (SEQ ID NO: 147)TTTACAGGTGAAGGTCACGTTGG 4. (SEQ ID NO: 148) GTAGCTAAATAAGGGTATGGAGG(reverse)

FIG. 2 presents the sequence (SEQ ID NO: 171) obtained from PCRamplification of the replaced DNA locus in transformed Chlamydomonasplastid DNA modified by the Edit Plasmid approach. Underlined sequencesat the 5′ and 3′ ends indicate wild-type chloroplast genomic sequencethat is not present on the Edit Plasmid. Sequences in bold font indicatethe short homologous regions present in the donor DNA on the EditPlasmid. Sequences that are both in bold font and underlined indicateguide RNA target sites present in the replaced DNA. The guide RNA targetsites in the donor DNA have been modified to prevent nuclease activityafter integration into the plastid genome. Sequences that have doubleunderlining indicate silent mutations at the 3′ side of guide RNA sitesto preclude re-cleavage by Cas9/sgRNA. The codon-optimized GFP codingregion is presented in italics. Homologous recombination occurred asexpected; i.e., there were no sequence changes either in the replacedDNA or in the surrounding wild-type plastid DNA.

The Edit Plasmids for Chlamydomonas chloroplasts were constructed asfollows. Polynucleotides encoding Cas9 and guide RNA were cloned intothe vector and were operably linked to appropriate promoters andterminators to allow for expression in chloroplasts. The vector waseither pBR322 or pUC19, each of which contained the replication originof pMB1 which previously was shown to replicate in chloroplasts (Boyntonet al. 1988 Science 240: 1534-1538).

The nucleic acid sequence (SEQ ID NO: 149) encoding SpCas9 (SEQ ID NO:150) was codon-optimized for Chlamydomonas chloroplast expression. Theoptimization was performed using a web-based Codon Usage Database(Nakamura et al. 2000 Nucleic Acids Res. 28: 292). The optimized genewas synthesized by GenScript (Piscataway, N.J.). The promoter used forCas9 gene expression was either the Chlamydomonas psaA-exon 1 promoterwith its 5′ UTR or the Chlamydomonas psbD promoter with its 5′ UTR (SEQID NO: 151 & SEQ ID NO: 152, respectively). The terminator used for Cas9gene expression was the rbcL 3′ UTR (SEQ ID NO: 153).

For expression of sgRNA, a tRNA promoter and its corresponding 3′ UTR(SEQ ID NO: 154 and SEQ ID NO: 155, respectively) were derived from theChlamydomonas plastid trnWgene locus. For the proper processing of sgRNAafter transcription, the endogenous chloroplast tRNA processing systemwas utilized as described in Xie et al. 2015 (Proc Natl Acad Sci USA112: 3570-3575). For example, for expression of one guide RNA, a sgRNAsequence was placed between two tRNAs. The configuration was“tRNA-1-sgRNA-tRNA-2”. For expression of two sgRNAs, the configurationwas “tRNA-1-sgRNA-1-tRNA-2-sgRNA-2-tRNA-3”. The following tRNA sequencesfrom Chlamydomonas plastid DNA: trnW (SEQ ID NO: 156), trnK (SEQ ID NO:157), and trnL (SEQ ID NO: 158) were employed.

A selectable marker expression cassette for the aadA coding region (SEQID NO: 159), to provide spectinomycin resistance, was also present onall the Edit Plasmid constructs. The promoter and terminator for theselectable marker expression cassette were the Chlamydomonas rbcLpromoter with its 5′ UTR (SEQ ID NO: 160) and the Chlamydomonas psbA 3′UTR (SEQ ID NO: 161), respectively. Plasmids that carried only a Cas9expression cassette and selectable marker expression cassette wereconstructed for use as controls.

For DNA replacement experiments, donor DNA was designed which consistedof a GFP coding region surrounded by homologous recombination regions.The GFP coding sequence (SEQ ID NO: 162) was designed to becodon-optimized for Chlamydomonas chloroplast gene expression accordingto the method of Franklin et al. 2002 (Plant J 30: 733-744). Forhomologous recombination of the donor DNA after double-strand breaks byCas9/double sgRNAs, we selected homologous regions of 74 or 76 bp each(HR1-HR4; SEQ ID NO: 163-SEQ ID NO: 166) from gRNA target sites in theChlamydomonas chloroplast gene, psaA-Exon 3. The short length (74 or 76bp) of each homologous sequence was chosen to minimize the occurrence ofendogenous homologous recombination without double-strand breaksmediated by Cas9/guide RNA (Dauvillee et al. 2004 PhotosynthesisResearch 79: 219-224). The configuration of the donor DNA with itscomponents is “1^(st) HR-GFP-2^(nd) HR”. The GFP sequence was derivedfrom Franklin et al. 2002 (Plant J. 30:733-744). To protect the donorDNA from further cleavage by Cas9 and to facilitate the Genome Sweepprocess, homologous recombination sequences also contained silentmutations at the target sites that precluded cleavage by Cas9 and guideRNAs. Homologous recombination was designed to give an in-frame fusionof GFP with the psaA gene product. Components in the Edit Plasmids forDNA replacement experiments included donor DNA as well as the Cas9,double sgRNAs and selectable marker expression cassettes described inthe previous section. The same vector backbone was used as in theprevious section, as well. As negative controls, plasmids lacking theCas9 expression cassette were used.

Tables 2 and 3 list the components of the constructs described in thissection.

TABLE 2 Components of Edit Plasmids for Chlamydomonas ChloroplastsConstruct Expr Cassette 1* Expr Cassette 2** Donor DNA YP5P_(psaA):Cas9co N/A N/A YP7 P_(psaA):Cas9co 1X-sgRNA-1 N/A YP8P_(psaA):Cas9co 1X-sgRNA-2 N/A YP9 P_(psaA):Cas9co 1X-sgRNA-3 N/A YP10P_(psaA):Cas9co 1X-sgRNA-4 N/A YP11 P_(psaA):Cas9co 2X-sgRNA-1 N/A YP12P_(psaA):Cas9co 2X-sgRNA-2 N/A YP13 P_(psaA):Cas9co 2X-sgRNA-1HR1:GFPco:HR2 YP14 P_(psaA):Cas9co 2X-sgRNA-2 HR3:GFPco:HR4 YP6P_(psbD):Cas9co N/A N/A YP15 P_(psbD):Cas9co 1X-sgRNA-1 N/A YP16P_(psbD):Cas9co 1X-sgRNA-2 N/A YP17 P_(psbD):Cas9co 1X-sgRNA-3 N/A YP18P_(psbD):Cas9co 1X-sgRNA-4 N/A YP19 P_(psbD):Cas9co 2X-sgRNA-1 N/A YP20P_(psbD):Cas9co 2X-sgRNA-2 N/A YP21 P_(psbD):Cas9co 2X-sgRNA-1HR1:GFPco:HR2 YP22 P_(psbD):Cas9co 2X-sgRNA-2 HR3:GFPco:HR4 YP23 N/A2X-sgRNA-1 HR1:GFPco:HR2 YP24 N/A 2X-sgRNA-2 HR3:GFPco:HR4 YP25P_(psaA):Cas9co 2X-sgRNA-1 HR1:GFPco:HR2 YP26 P_(psaA):Cas9co 2X-sgRNA-2HR3:GFPco:HR4 YP27 P_(psbD):Cas9co 2X-sgRNA-1 HR1:GFPco:HR2 YP28P_(psbD):Cas9co 2X-sgRNA-2 HR3:GFPco:HR4 YP29 N/A 2X-sgRNA-1HR1:GFPco:HR2 YP30 N/A 2X-sgRNA-2 HR3:GFPco:HR4 YP31 P_(psaA):Cas9co2X-sgRNA-1 N/A YP32 P_(psaA):Cas9co 2X-sgRNA-2 N/A YP33 P_(psbD):Cas9co2X-sgRNA-1 N/A YP34 P_(psbD):Cas9co 2X-sgRNA-2 N/A *Each ExpressionCassette 1 used the rbcL terminator. **Each Expression Cassette 2encoded either one (1X) or two (2X) guide RNAs.

TABLE 3 Components of Expression Cassette 2 Encoding One or Two GuideRNAs Name Component Detail* 1X-sgRNA-1 trnW-sgRNA591-trnK 1X-sgRNA-2trnW-sgRNA717-trnK 1X-sgRNA-3 trnW-sgRNA747-trnK 1X-sgRNA-4trnW-sgRNA843-trnK 2X-sgRNA-1 trnW-sgRNA591-trnK-sgRNA717-trnL2X-sgRNA-2 trnW-sgRNA747-trnK-sgRNA843-trnL *Each Expression Cassette 2used both the trnW promoter and trnW terminator.

Edit Plasmids were transformed into wild-type Chlamydomonas (CC-125)according to the methods of Barrera et al. 2014 (Methods Mol. Biol.1132: 391-399) and Ramesh et al. 2011 (Methods Mol. Biol. 684: 313-320).Chloroplast transformants were selected using Tris-Acetate-Phosphate(TAP) media supplemented with 100 μg/ml of Spectinomycin.

To assess DNA replacement events, we transformed Edit Plasmid YP13containing donor DNA into CC-125 (wild-type Chlamydomonas reinhardtii)and randomly selected spectinomycin-resistant colonies. Controlconstruct was YP23. Pooled transformed cell lines were used to preparechloroplast DNAs according to Barrera et al. 2014 (Methods Mol. Biol.1132: 391-399). Pool size for YP13 was 20 independent colonies and thepool size for YP23 was 16 independent colonies. For PCR amplification ofthe targeted recombination region, we used primer sets which consistedof a chloroplast genomic region-specific primer and a GFP gene-specificprimer. Primer Set 1 (PS1) was designed to amplify the 5′ end of GFPintegration region while Primer Set 2 (PS2) was designed to amplify the3′ end.

1. PS1 FWD Primer (SEQ ID NO: 167) GCTGGTTGGTTCCACTACCAC2. PS1 REV Primer (SEQ ID NO: 168) CACCTTCAAATTTTACTTCAGCACGTG3. PS2 FWD Primer (SEQ ID NO: 169) CATACGGTGTACAATGTTTCAGTCG4. PS2 REV Primer (SEQ ID NO: 170) GTGAGAAATAATAGCATCACGGTGAC

The primer sets were designed to avoid amplification of wild-typechloroplast genome or of the Edit Plasmid. Using the above primer sets,the expected size of each amplicon is the following: 852 bp for PrimerSet 1 and 712 bp for Primer Set 2. After PCR amplification, wesuccessfully obtained amplicons of the expected sizes from twoindependent pools of Chlamydomonas cell lines transformed with YP13. Thecorresponding DNA fragments were not amplified from YP23, the controlconstruct without the Cas9 expression cassette.

We sequenced the amplified DNA fragments to confirm successful DNAreplacement through Cas9 activity. We obtained the sequence encompassingthe donor DNA locus in the transformed Chlamydomonas chloroplast DNA(see FIG. 2) (SEQ ID NO: 171). The genomic sequence corresponded to theexpected sequence from insertion of the donor DNA at the two Cas9cleavage sites. As seen in FIG. 2, the replaced DNA contained the twomodified guide RNA target sites in the psaA gene that were encoded inthe donor DNA. Additionally, the 3-nt PAM sequence is no longer presentadjacent to each target sequence, corresponding to the exact sequence ofthe donor DNA. Also shown in FIG. 2 are sequences at the 5′ and 3′ ends(shown with underlining) that are wild-type chloroplast genomicsequences not present on the Edit Plasmid, which are contiguous to theHR regions (shown in bold font) present in the Edit Plasmid. In summary,DNA replacement was observed in Chlamydomonas chloroplasts exactly asdesigned by use of an Edit Plasmid that encoded a Cas9 expressioncassette, a multiple guide RNA expression cassette and a donor DNAtemplate.

Once a chloroplast DNA site is cleaved by Cas9, DNA repair should berecognizable by the presence of any of the following: nucleotidesubstitution, small insertion or small deletion. We analyzedspectinomycin-resistant colonies transformed with YP11 and YP31 EditPlasmid constructs for evidence of such DNA repair. We included YP29,the construct without the Cas9 expression cassette, as a control. Toenrich for edited events, we utilized the presence of the Availrecognition sequence (GGWCC where W is either A or T) at one of theCas9/gRNA cleavage sites (SEQ ID NO: 146, CTTCACCTGTAAATGGACCACGG).First, we extracted DNA from randomly selected Chlamydomonas colonies(15 colonies from YP11 transformants, 10 colonies from YP31transformants, and five colonies from YP29 transformants). We thenpooled extracted DNA for Q5® high-fidelity polymerase-based PCRamplification (New England BioLabs) of the genomic region containing thetarget site (one pool contained DNA from five colonies). We used thefollowing primers: PS1 FWD Primer (SEQ ID NO: 167) and PS2 REV Primer(SEQ ID NO: 170). Amplified DNA products were purified and subjected toAvaII digestion overnight. After gel-electrophoresis, the regioncorresponding to 700-900 bp of each pool, containing undigested DNA of795 bp, was cut out of an agarose gel and the DNA was extracted.Extracted DNA was then directly cloned into pMiniT2.0 vector accordingto a manufacturer's protocol (New England BioLabs, Ipswich, Mass.). Werandomly selected twelve E. coli colonies from each pool of YP11 andYP31 transformants and eight colonies from the control YP29 pool andperformed PCR amplification using the same primer pair, PS1 FWD Primerand PS2 REV Primer. Aliquots of PCR reactions were digested again withAvail to further select candidates for DNA repair events. One each fromtwo pools of YP11 constructs, one from one pool of YP31 transformants,four from the other pool of YP31 transformants and three from the YP29transformants were identified and subjected to Sanger-sequencing todeduce the nucleotide composition of each candidate clone. In addition,we included PCR amplicons of 15 randomly selected colonies from the YP29control pool for sequencing. Analysis of sequencing results showed thattwo transformants of YP11 and two of YP31, each from a different pool,had a single nucleotide substitution at the target sites. We observedthe following two types of substitution: G to A, resulting in GAACC; andA to G, resulting in GGGCC; relative to the wild-type sequence, GGACC.Each of these two changes were detected in transformants from eachconstruct, YP11 and YP31; however, none of the sequenced clones from thecontrol YP29 transformants showed any change at the target site (i.e.,each control transformant retained the Avail site). In summary, we haveshown that four independent nucleotide substitution events have occurredat a guide RNA target site, consistent with cleavage by Cas9 andsubsequent DNA repair in the chloroplast.

That which is claimed:
 1. A method for altering the genome of anorganelle, the method comprising: a. introducing into an organelle arecombinant DNA construct comprising the following: i. a firstpolynucleotide encoding at least one guide RNA, wherein the at least oneguide RNA directs a polynucleotide guided polypeptide to cleave at leastone target sequence present in an organelle genome; ii. a secondpolynucleotide encoding a polynucleotide guided polypeptide, wherein thepolynucleotide guided polypeptide, when associated with the guide RNA,cleaves the at least one target sequence; iii. optionally, a thirdpolynucleotide encoding at least one homologous organelle DNA sequence,wherein the at least one homologous organelle DNA is of sufficient sizefor homologous recombination, wherein integration of the at least onehomologous organelle DNA sequence into the organelle genome results inremoval of the at least one target sequence; iv. optionally, a fourthpolynucleotide encoding at least one selectable marker or at least onescreenable marker, or both; wherein the fourth polynucleotide isoperably linked to a promoter that is functional in the organelle; andv. optionally, a fifth polynucleotide encoding an origin of replicationthat is functional in the organelle; and b. growing a cell comprisingthe organelle of (a) under conditions in which the first polynucleotideof (i) and the second polynucleotide of (ii) are each expressed.
 2. Themethod of claim 1, wherein the method further comprises: (c) selecting acell having an organelle that comprises an altered genome.
 3. The methodof claim 2, wherein the method further comprises: (d) selecting a cellthat is homoplasmic for the altered genome of the organelle.
 4. Themethod of claim 3, wherein the organelle is a plastid.
 5. The method ofclaim 3, wherein the organelle is a mitochondrion.
 6. The method ofclaim 1, comprising the third polynucleotide of (iii), wherein the thirdpolynucleotide of (iii) comprises a sixth and a seventh polynucleotide,wherein the sixth and the seventh polynucleotides correspond to twoadjacent regions of homology in the organelle genome, wherein the sixthand seventh polynucleotides are separated by a sequence that isheterologous to the organelle DNA.
 7. The method of claim 6, wherein thesequence that is heterologous to the organelle DNA comprises at leastone selected from the group consisting of: the first polynucleotide, thesecond polynucleotide, the fourth polynucleotide an eighthpolynucleotide, and any combination thereof, wherein the eighthpolynucleotide encodes an RNA that is heterologous to the organelle. 8.The method of claim 1, wherein the at least one guide RNA is present ona polycistronic transcription unit.
 9. The method of claim 8, whereinthe at least one guide RNA is processed from a polycistronic RNA aftertranscription of the polycistronic transcription unit by use of at leastone selected from the group consisting of: an RNA cleavage site, a Csy4cleavage site, a ribozyme cleavage site, a polynucleotide guidedpolypeptide cleavage site, the presence of a tRNA sequence, and anycombination thereof.
 10. The method of claim 9, wherein thepolycistronic RNA comprises a first tRNA sequence 5′ to the at least oneguide RNA and a second tRNA sequence 3′ to the at least one guide RNA.11. The method of claim 6, wherein at least one selected from the groupconsisting of: the first polynucleotide, the second polynucleotide, thefourth polynucleotide, the fifth polynucleotide, and any combinationthereof, is located outside the region bounded by the sixth and theseventh polynucleotide.
 12. The method of claim 6, comprising the fourthand the fifth polynucleotides, wherein both the fourth and the fifthpolynucleotides are located outside the region bounded by the sixth andthe seventh polynucleotides.
 13. The method of claim 6, wherein saidpolynucleotide-guided polypeptide is selected from the group consistingof: a Cas9 protein, a MAD2 protein, a MAD7 protein, a CRISPR nuclease, anuclease domain of a Cas protein, a Cpf1 protein, an Argonaute, modifiedversions thereof, and any combination thereof.
 14. The method of claim6, wherein the method further comprises introducing into the organelle apolynucleotide encoding at least one selectable marker selected from thegroup consisting of: a positive selectable marker, a negative selectablemarker, and any combination thereof.
 15. The method of claim 14, whereinthe method further involves growing the cell in the presence of apositive selection agent and selecting a cell that is homoplasmic forthe altered genome of the organelle.
 16. The method of claim 15, whereinthe method further involves growing the cell in the absence of thepositive selection agent, followed by selecting a cell that lacks anon-integrated recombinant DNA construct.
 17. The method of claim 15,wherein the method further involves growing the cell in the absence ofthe positive selection agent, followed by growing the cell in thepresence of a negative selection agent, followed by selecting a cellthat lacks a non-integrated recombinant DNA construct.
 18. The method ofclaim 6, wherein the cell is a plant cell, wherein the organelle is aplastid or a mitochondrion, and wherein the method further comprisesregenerating a plant from the plant cell comprising an altered organellegenome.
 19. The method of claim 6, wherein the cell is a yeast cell oran algal cell.
 20. A recombinant DNA construct comprising the following:i. a first polynucleotide encoding at least one guide RNA, wherein theat least one guide RNA directs a polynucleotide guided polypeptide tocleave at least one target sequence present in an organelle genome; ii.a second polynucleotide encoding a polynucleotide guided polypeptide,wherein the polynucleotide guided polypeptide, when associated with theguide RNA, cleaves the at least one target sequence; iii. a thirdpolynucleotide comprising a sixth and a seventh polynucleotide, whereinthe sixth and the seventh polynucleotides correspond to two adjacentregions of homology in the organelle genome, wherein the sixth andseventh polynucleotides are separated by a sequence that is heterologousto the organelle DNA, wherein the sequence that is heterologous to theorganelle DNA comprises at least one selected from the group consistingof: the first polynucleotide, the second polynucleotide, the fourthpolynucleotide, an eighth polynucleotide, and any combination thereof,wherein the eighth polynucleotide encodes an RNA that is heterologous tothe organelle; iv. optionally, a fourth polynucleotide encoding at leastone selectable marker or at least one screenable marker, or both;wherein the fourth polynucleotide is operably linked to a promoter thatis functional in the organelle; and v. optionally, a fifthpolynucleotide encoding an origin of replication that is functional inthe organelle.
 21. A yeast cell, algal cell, plant cell, plant, seed,root, stem, leaf, flower, fruit, or bean comprising the recombinant DNAconstruct of claim
 20. 22. A method for altering the genome of anorganelle, the method comprising: a. introducing into a cell: i. apolynucleotide encoding an RNA sequence comprising an organelletargeting RNA operably linked to a guide polynucleic acid, wherein theguide polynucleic acid directs a polynucleotide guided polypeptide tocleave a target sequence present in an organelle genome, wherein thepolynucleotide is operably linked to at least one regulatory element;and either ii. a second polynucleotide encoding a modifiedpolynucleotide guided polypeptide, wherein the second polynucleotide isoperably linked to at least one regulatory element, and wherein themodified polynucleotide guided polypeptide comprises a polynucleotideguided polypeptide operably linked to an organelle targeting peptide;wherein the organelle targeting RNA of (i) and the organelle targetingpeptide of (ii) each target the same organelle; or iii. a thirdpolynucleotide, wherein the third polynucleotide is operably linked toat least one regulatory element, wherein the third polynucleotideencodes an RNA molecule comprising an organelle targeting RNA operablylinked to an RNA sequence encoding a polynucleotide guided polypeptide;wherein the organelle targeting RNA of (i) and the organelle targetingRNA of (iii) each target the same organelle; and b. growing the cellunder conditions in which the polynucleotide of (i) and the secondpolynucleotide of (ii) or the third polynucleotide of (iii) are bothexpressed.
 23. The method of claim 22, further comprising introducing apolynucleotide comprising at least one donor polynucleotide into theorganelle, wherein the at least one donor polynucleotide comprises atleast one homologous sequence with respect to the organelle genome,wherein integration of all or part of the at least one donorpolynucleotide into the organelle genome results in removal of thetarget site of the guide polynucleic acid.
 24. A method for altering agenome of an organelle, the method comprising: (a) introducing into anorganelle of a cell: (i) at least one guide RNA, wherein the at leastone guide RNA directs a polynucleotide guided polypeptide to cleave atleast one target sequence present in the genome of the organelle; (ii) apolynucleotide guided polypeptide, wherein the polynucleotide guidedpolypeptide, when associated with the at least one guide RNA, cleavesthe at least one target sequence; and (iii) a replacement DNA; and (b)selecting a cell comprising an organelle comprising the replacement DNA.25. The method of claim 24, wherein the replacement DNA of step (a) part(iii) comprises fragments of organellar DNA or a complete organellar DNAfrom a cultivar, line, sub-species and other species and is distinctfrom the genome of the organelle of step (a).
 26. The method of claim24, wherein the at least one target sequence is not present in thereplacement DNA.
 27. The method of claim 24, wherein after step (a) part(ii) and prior to step (a) part (iii), a cell is selected in which thegenome of the organelle has been eliminated.